J Proteome Res. 2016 Mar 4;15(3):1051-8.

High-Throughput LC-MS/MS Method for Direct Quantification of Glucuronidated, Sulfated, and Free Enterolactone in Human Plasma.

Nørskov NP1, Kyrø C2, Olsen A2, Tjønneland A2, Bach Knudsen KE1.
  • 1Aarhus University , Department of Animal Science, AU-Foulum, Blichers Alle 20, P.O. Box 50, DK-8830 Tjele, Denmark.
  • 2Danish Cancer Society Research Center , Strandboulevarden 49, DK-2100 Copenhagen, Denmark.



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: LC−MS/MS; enterolactone glucuronide; enterolactone sulfate; free enterolactone; human plasma; method

PMID: 26809233



Enterolactone is the main mammalian lignan coming from the metabolism of plant lignans in the gastrointestinal tract of humans. Plant lignans is a group of compounds that can be found in many different plant derived foods consumed by humans; fruits, vegetables, bread, flaxseed, nuts and etc1,2. 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 health3,4,5,6. Therefore, it was hypothesized that plant lignans and especially their metabolite enterolactone, is one the contributors to the positive health effects after consumption of foods high in dietary fiber. The positive health effects of enterolactone have been related to the estrogen-like structure with biological activities ranging from antioxidative to estrogenic/antiestrogenic7. Many studies have been performed trying to identify the role of enterolactone in breast, prostate, and colon cancers, as well as in type 2 diabetes and cardiovascular diseases. However, results from these studies have been variable and difficult to explain since the effects of enterolactone have been both positive and negative7. An important aspect to consider is the fact that all these studies have been performed with enterolactone estimated as total enterolactone/ free enterolactone (Figure 1.), not taking into account that enterolactone exists mainly as enterolactone glucuronide (Figure 2.) and sulfate (Figure 3.) in the human body. The lack of analytical method to measure enterolactone as glucuronide and sulfate was the main reason why these studies were performed with free form of enterolactone. Therefore our purpose was to develop a method that could measure three intact forms of enterolactone in human blood; free enterolactone, enterolactone glucuronide and sulfate. Moreover, we wanted the method to be rapid and easy to perform with high sensitivity over a wide concentration range.




Liquid Chromatography-Mass Spectrometry (LC-MS/MS) is a very sensitive technology and does not require tedious sample preparation prior to measurements compared to Gas Chromatography-Mass Spectrometry (GC-MS) and fluoroimmunoassay. The time spend on sample preparation and measurement is an issue when analyzing thousands of samples. Using our method it is possible to measure approximately 200 samples a day compared to fluoroimmunoassay where the same number of samples takes one week.

Another important aspect of this method is that the method was developed to measure intact forms of enterolactone as glucuronide, sulfate and free enterolactone. Using authentic standards of enterolactone, enterolactone glucuronide and enterolactone sulfate we were able to optimize the MS instrument to the most sensitive transition with low limits of quantification (LLOQ) of 16 pM for enterolactone sulfate, 26 pM for enterolactone glucuronide and 86 pM for free enterolactone. Chromatographic separation was optimized to 2.6 minutes per sample allowing to run high number of samples per day (Figure 4).



fig4Figure 4. Typical MRM chromatogram of authentic standards in concentration 1.56 ng/mL. Enterolactone glucuronide RT (1.33), Enterolactone sulfate RT (1.38), Internal standard RT (1.51), and Enterolactone RT (1.65).


With the developed method we determined the percentage distribution of the enterolactone glucuronide, sulfate and free enterolactone in 1961 and 1952 blood samples from men and women, respectively. This has not been done before on such a high number of samples. Moreover, we were interested to look into whether the percentage distribution was similar in men and women. Our results showed that by far the most abundant form of enterolactone is glucuronidated accounting for approximately 95 % of the total concentration of enterolactone in the blood followed by enterolactone sulfate (app. 5 %) and free enterolactone was quantifiable in only few samples. We did not observe any difference between men and women. Whether enterolactone play any significate role in the protection against breast cancer or any other cancer is still to be answered. However, we hope that by using this analytical method we can come closer to answering this question.

The importance of this novel analytical 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 studies where the number of samples is very high.



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Acknowledgment: We thank Innovation Fund Denmark for financing the project “The effects of enterolignans in chronic diseases – ELIN” (0603-00580B).



Natalja P. Nørskov, Ph.D.

Postdoc at the Department of Animal Science

Aarhus University

Blichers Alle 20

8830 Tjele

Phone: +4587157967

Fax: +4587157724

E-mail: Natalja.Norskov@anis.au.dk





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