MSACL 2017 EU Abstract

Application of LC-MS/MS Metabolomics - Quantitative High-Throughput Method for Direct Measurements of Enterolactone Glucuronide, Sulfate and Free Enterolactone

Natalja Pustovalova Nørskov (Presenter)
Aarhus University

Bio: Natalja P. Nørskov has completed her PhD at the age of 37 years from Aarhus University (Denmark). After her PhD she continued her work as a postdoc at Aarhus University working on development of high throughput methods for quantitative metabolomics using LC-MS instruments. Many of the methods are now published in the international journals. She is also a leader of a PhD course titled, “Hands-on targeted and untargeted LC-MS metabolomics with emphasis on measuring phytochemicals in plasma and urine”.

Authorship: Natalja P. Nørskov(1), Cecilie Kyrø(2), Anja Olsen(2), AnneTjønneland(2), and Knud Erik Bach Knudsen(1)
(1)Aarhus University, Department of Animal Science, AU-Foulum, Blichers Alle 20, P.O. box 50 DK-8830 Tjele, Denmark (2)Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen, Denmark

Short Abstract

Enterolactone was discovered more than 30 years ago and since then it has been considered as a biomarker of healthy lifestyle in epidemiological studies. To measure enterolactone in plasma requires enzymatic hydrolyses as enterolactone is typically conjugated with glucuronic acid and sulfate. We have developed and validated a high-throughput LC-MS/MS method to quantify enterolactone without the need for hydrolysis. This has several advantages such as simple sample preparation procedure and measurement of enterolactone in its intact forms. The method has short chromatographic run time of 2.6 min with good accuracy and precision and high sensitivity, LLOQ down to 16 pM.

Long Abstract


Enerolactone was discovered more than 30 years ago and since then it has been considered as a biomarker of healthy lifestyle in epidemiological studies. Enterolactone is the main mammalian lignan coming from the metabolism of plant lignans in the gastrointestinal tract of humans. Plant lignans are non-nutritive compounds found in many different plant foods consumed by humans; fruits, vegetables, bread, flaxseed, nuts and etc.(1,2). Epidemiological studies have indicated that diets high in dietary fiber, containing among other things lignans, are associated with positive nutritional and physiological effects with importance to human health. Therefore, it was hypothesized that plant lignans and especially their metabolite enterolactone, is one the contributors to the positive health effects. It was further hypothesized that enterolactone may play a preventive role in lifestyle diseases such as type 2 diabetes and and cardiovascular diseases. Moreover, due to its aromatic structure, enterolactone has similarities to steroid hormones and therefore has been considered to be protective against hormone-dependent cancers (3,4,5). Many studies have been performed trying to identify the role of enterolactone in breast, prostate, and colon cancers. However, results from these studies have been variable and difficult to explain, since the effect of enterolactone as a weak estrogen has been shown to be both agonistic and antagonistic (6). An important aspect to consider is the fact that all these studies have been performed with enterolactone measured as total enterolactone, not taking into account the conjugation pattern of enterolactone. To measure enterolactone in human plasma requires enzymatic hydrolyses with enzymes β-glucuronidase and sulfatase, as enterolactone is typically conjugated with glucuronic acid and sulfate.


We have developed and validated a high-throughput LC-MS/MS method to quantify enterolactone without the need for hydrolysis with advantage to measure enterolactone in its intact forms. This method is 5 times faster than fluoroimmunoassay due to simple sample preparation procedure and short chromatographic run time of only 2.6 min. The method has shown good accuracy and precision and very high sensitivity, with LLOQ for enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM and free enterolactone at 86 pM. We used the authentic standards of enterolacrone, enterolactone glucuronide, and enterolactone sulfate to optimize LC-MS/MS conditions (Q1 and Q3, source and chromatographic separation). Further the samples were cleaned-up by Solid Phase Extraction (SPE) and the method was validated using test plasma spiked with three different concentrations of standards: low, medium, and high. Accuracy, precision, and recovery were determined for each analyte at three concentrations, mentioned above. Recovery of enterolactone glucuronide varied from 86 – 100 %, enterolactone 99 -111 % and enterolactone sulfate 66 – 101 % depending on the concentration tested. Accuracy was calculated as relative error (ER) between true and measured values. Precision was calculated as relative standard deviation (RSD). The acceptance criterion for both accuracy and precision was that they should not exceed by more than 15 %.


We have adapted the method to measure enterolactone and its conjugates in plasma samples from almost 6000 participants from the Danish Diet, Cancer and Health cohort (7). The participants were all healthy when the blood was drawn at baseline, but were later diagnosed with the one or more of the following diseases: breast cancer, prostate cancer, colorectal cancer, type 2 diabetes, or myocardial infraction. From baseline, data on lifestyle and dietary habits were available. The interrelationship between these factors and enterolactone conjugates, total enterolactone and ratio (enterolactone sulfate/total concentration of enterolactone) was investigated. Multivariate data analysis was performed using an unsupervised method for pattern recognition Principal Component Analysis (PCA). We included the following baseline lifestyle and dietary variables: smoking status (former, current, never), alcohol use (g/day), age, BMI (kg/m2), participation in sport (yes/no), whole-grain intake (g/day), intake of fruits (g/day), intake of vegetables (g/day) and schooling (low, middle, high). Further, information on which diseases type (breast, prostate or colorectal cancers, diabetes and myocardial infraction) the participants were diagnosed with. The results of PCA indicated that the total concentration of enterolactone and the concentration of enterolactone glucuronide and sulfate positively correlated to sport, schooling, fruits-, vegetables- and whole-grain intake and negatively to ratio of enterolactone, BMI, disease type, smoking status, alcohol intake and age. That was also expected since people who eat more fruits, vegetables and whole-grain have higher enterolactone concentration. Interestingly, the ratio of enterolactone correlated positively to age, BMI and disease type. Since the ratio describes the conjugation pattern of enterolactone it can be proposed as a better measure of metabolic modifications influenced by age, BMI and disease than the total concentration of enterolactone and concentration of enterolactone glucuronide and sulfate. For example the activity of glucuronidation catalyzing enzyme, uridine diphosphate glucuronosyltransferases (UDPGT) has been previously shown to be affected by genetic polymorphism as well as dietary intake, age, gender, BMI and cigarette smoking (8,9). Therefore the conjugation pattern of enterolactone may depend on the activity of glucuronidation- and sulfation catalyzing enzymes as well as availability of their substrates and therefore be affected by metabolic changes. In general PCA loading plots for women and men were similar. However there were also some differences with regard to age and alcohol intake. In PCA plot for woman “age” was more correlated with BMI, ratio of enterolactone and disease type, whereas for man “age” was more an independent variable. Oppositely, “alcohol intake” for woman was more an independent variable, whereas for man “alcohol intake” correlated more with smoking status. Moreover, we found enterolactone glucuronide to be the major conjugation form representing approximately 95 % of total enterolactone in both man and woman. To our knowledge our study is the first to demonstrate the conjugation pattern of enterolactone in such a high number of samples.

Conclusions & Discussion

The importance of this novel, targeted LC-MS/MS method is two-fold. Firstly, we hope that measuring enterolactone in its intact forms will contribute with new knowledge on the role of enterolactone in human health. Secondly, since the method is rapid and easy to perform it can be used for high-throughput of samples and therefore will be the method of choice for future epidemiological investigations and clinical diagnosis.

References & Acknowledgements:


1. Axelson, M.; Sjovall, J.; Gustafsson, B. E.; Setchell, K. D. R., ORIGIN OF LIGNANS IN MAMMALS AND IDENTIFICATION OF A PRECURSOR FROM PLANTS. Nat. 1982, 298, 659-660.

2. Setchell, K. D. R.; Lawson, A. M.; Borriello, S. P.; Harkness, R.; Gordon, H.; Morgan, D. M. L.; Kirk, D. N.; Adlercreutz, H.; Anderson, L. C.; Axelson, M., LIGNAN FORMATION IN MAN - MICROBIAL INVOLVEMENT AND POSSIBLE ROLES IN RELATION TO CANCER. Lan. 1981, 2, 4-7.

3. Fardet, A., New hypotheses for the health-protective mechanisms of whole-grain cereals: what is beyond fibre? Nutrition Research Reviews 2010, 23, 65-134.

4. de Munter, J. S.; Hu, F. B.; Spiegelman, D.; Franz, M.; van Dam, R. M., Whole grain, bran, and germ intake and risk of type 2 diabetes: A prospective cohort study and systematic review. Plos Medicine 2007, 4, 1385-1395.

5. Slavin, J., Whole grains and human health. Nutrition Research Reviews 2004, 17, 99-110.

6. Adlercreutz, H., Lignans and human health. Crit. Rev.Clin. Lab. Sci. 2007, 44, 483-525.

7. Olsen, A.; Knudsen, K. E. B.; Thomsen, B. L.; Loft, S.; Stripp, C.; Overvad, K.; Moller, S.; Tjonneland, A., Plasma enterolactone and breast cancer incidence by estrogen receptor status. Cancer Epidemiol. Biomark. Prev. 2004, 13, 2084-2089.

8. Liston, H. L.; Markowitz, J. S.; DeVane, C. L., Drug glucuronidation in clinical psychopharmacology. Journal of Clinical Psychopharmacology 2001, 21, 500-515.

9. Burchell, B.; Lockley, D.; Fettes, M., Genetic variation of UDP-glucuronosyltransferases and drug toxicity. Toxicology 2003, 192, 56-57.


We thank Innovation Fund Denmark for financing the project “The effects of enterolignans in chronic diseases - ELIN” (0603-00580B). The authors have no conflict of interest.

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