Isoflavone content in selected legumes (mg/100 g, edible portion—the mean value derived from multiple experiments) [12].
Abstract
Phytoestrogens are natural compounds found in various plant species and they have the ability to bind to the estrogenic receptors, exerting agonist and/or antagonist effects. The main classes of phytoestrogens are isoflavones, lignans, and coumestranes. Isoflavones are plant bioactive nonsteroidal polyphenolic metabolites with antioxidant properties. They have a very close structure with 17β-estradiol and possess estrogenic/antiestrogenic effects. The main dietary source of isoflavones is soy (Glycine max L.). Other legumes, such as red clover (Trifolium pratense L.), alfalfa (Medicago sativa L.), and Genista species, have important content in isoflavones, showing nutritional or phytotherapeutic interest. In plants, isoflavones can be found mainly as non-active glycosides which are converted after ingestion, in the corresponding aglycones (e.g., genistein, daidzein) that have pharmacological activity. Many studies have demonstrated the benefits of dietary isoflavones in menopause and multiple chronic pathologies, including cardiovascular diseases, osteoporosis, and hormonal cancers. Dietary intake of isoflavones is widespread, mainly due to the consumption of soybean products. Analytical methods applied for the quantification of isoflavones allow both assessment of dietary intake of isoflavones and highlighting natural sources with phytotherapeutic potential. Health benefits of isoflavones justify the interest for this class of functional food; therefore, further clinical and epidemiological studies are required.
Keywords
- nutraceuticals
- phytoestrogens
- isoflavones
- vegetables
- analysis
1. Introduction
Phytoestrogens are natural nonsteroidal compounds able to bind to estrogenic receptors and have both estrogenic and antiestrogenic activities. They are widespread in the plant kingdom being considered ubiquitous. The main classes of phytoestrogens are isoflavones, coumestans, and lignans.
Isoflavones are plant-derived secondary metabolites with a polyphenolic structure and antioxidant properties [1]. They pertain to the flavonoid class and are found mostly in plants belonging to Fabaceae family. Soy (
Several epidemiological studies have demonstrated the benefits of dietary isoflavones in menopause and multiple chronic pathologies, including cardiovascular diseases, osteoporosis, and hormonal cancers. The main mechanisms of action of isoflavones, their benefits to human health, and the factors involved in the modulation of their bioactivity are shown in this chapter. Moreover, the analytical methods used for their quantification in plant and food samples are introduced. These are very important methods to evaluate the human exposure to isoflavones and also to assess the optimum intake for human well-being.
2. Characteristics of isoflavones
2.1. Chemistry and metabolism of isoflavones
Isoflavones (IFs) are yellow pigments derived from 3-phenyl-benzopyrone (3-phenyl-chromone) structure. They are found in plants mostly as biologically inactive glycosides: 7-
The absorption of aglycones is fast and efficient. Plasmatic isoflavone levels increase up to micromolar-level values after the consumption of soy-based foods, compared to the nanomolar (≤40 nm) levels found in diets without soy [4]. First pharmacokinetic study on isolated and purified isoflavones was performed, when a single dose of 50 mg of aglycone or the equivalent dose of
Formononetin and biochanin A can be transformed to daidzein and genistein, respectively, through 4′-
Gut microbiota play a very important role in the isoflavone metabolism. The positive effects of a soy-rich diet derive from the existence of microorganisms in the gut capable of intense metabolization of isoflavones. It is the so-called equol producer phenotype, responsible for metabolizing daidzein to equol and identified through the equol/daidzein ratio in the 24-hour urine. Asian people (Japanese, Korean, or Chinese) and Western adult vegetarians are 50–60% equol producers, but equol producers are only 25–30% in Western population. This phenotype is rather stable and cannot be modulated through prebiotic or probiotic nutritional interventions [8]. Otherwise, there are differences between human and animal metabolism, and therefore in vivo results are not relevant to humans [9]. All tested animals had equol in urine after the ingestion of soy or clover [8]. Notably in rodents, equol constitutes 70–90% from the serum isoflavones, compared to humans where only 30% of the daidzein absorbed is metabolized as equol [4].
2.2. Isoflavone content in different sources
Isoflavones can be found in legumes [10–12], nuts, and some fruits, such as currants and raisins [13], coffee [14], and cereals [15], but the most important dietary sources are soybeans and their by-products [10, 12]. The content of isoflavones in several plants and foods is presented in Tables 1 and 2. Soy can be ingested as textured soy protein, as soy milk or drink, added to many fortified foods (e.g., energized bars, cereals, baby formula), or consumed as fermented soybean products, such as miso, natto, and tempeh (Table 3) [12]. Also, many food supplements containing soy isoflavones are on the market [16].
Food description | Daidzein | Genistein | Glycitein | Total IFs |
---|---|---|---|---|
Soybeans, green, mature seeds, raw | 61.70 | 60.07 | 7.07 | 128.83 |
Soybeans, mature seeds, raw (the mean values from Australia, Brazil, China, Europe, Japan, Korea, Taiwan, the USA) | 62.07 (27.77–78.86) | 80.99 (39.78–89.32) | 14.99 (9.01–22.37) | 154.53 (85.68–178.81) |
Soybeans, mature seeds, cooked, boiled | 30.76 | 31.26 | 3.75 | 65.11 |
Beans, common, raw ( | 0.29 | 0.30 | 0.00 | 0.59 |
Beans, adzuki, mature seeds, raw | 0.36 | 0.23 | 0.00 | 0.59 |
Beans, pinto, mature seeds, raw | 0.01 | 0.17 | - | 0.18 |
Black bean, sauce | 5.96 | 4.04 | 0.53 | 10.26 |
Chickpeas, mature seeds, raw | 0.21 | 0.06 | 0.18 | 0.38 |
Chickpeas, mature seeds, cooked, boiled | 0.00 | 0.02 | - | 0.02 |
Peas, green, split, mature seeds, raw | 0.32 | 0.11 | 0.00 | 0.44 |
Food description | Coumestrol | Formononetin | Biochanin A |
---|---|---|---|
Egg, whole, raw, fresh | 0.00 | 0.05 | 0.05 |
Alfalfa seeds, sprouted, raw | 1.60 | 1.43 | 0.04 |
Clover sprouts, raw | 14.08 | 3.15 | 0.59 |
Red clover | 1322.00 | 833.00 | - |
Soybeans, mature seeds, raw | 0.02 | 8.46 | 0.00 |
Soybeans, mature seeds, sprouted, raw | 0.34 | 0.03 | 0.00 |
Lima beans, large, mature seeds, raw | 0.14 | 0.32 | 0.27 |
Lima beans, large, mature seeds, boiled | 0.00 | 0.01 | 0.00 |
Chickpeas, mature seeds, raw | 0.01 | 0.12 | 1.54 |
Chickpeas, mature seeds, canned | 0.00 | 0.00 | - |
Food description | Daidzein | Genistein | Glycitein | Total IFs |
---|---|---|---|---|
Miso | 16.43 | 23.24 | 3.00 | 41.45 |
Natto | 33.22 | 37.66 | 10.55 | 82.29 |
Tempeh | 22.66 | 36.15 | 3.82 | 60.61 |
Tofu, raw, regular, prepared with calcium sulfate | 8.56 | 12.99 | 1.98 | 22.73 |
Soybeans, green, raw (includes edamame) | 20.34 | 22.57 | 7.57 | 48.95 |
Soybeans, green, cooked, boiled, drained, without salt (includes edamame) | 7.41 | 7.06 | 4.60 | 17.92 |
Soybeans, mature seeds, sprouted, raw | 12.86 | 18.77 | 2.88 | 34.39 |
Instant beverage, soy, powder, not reconstituted | 40.07 | 62.18 | 10.90 | 109.51 |
Soy cheese, unspecified | 5.79 | 11.14 | - | 25.72 |
Soy drink | 2.75 | 5.10 | - | 7.85 |
Soy flour (textured) | 67.69 | 89.42 | 20.02 | 172.55 |
Soy meal, defatted, raw | 80.77 | 114.71 | 16.12 | 209.58 |
Soy protein drink | 27.98 | 42.91 | 10.76 | 81.65 |
Soy protein isolate | 30.81 | 57.28 | 8.54 | 91.05 |
Soy yogurt | 13.77 | 16.59 | 2.80 | 33.17 |
Isoflavone content in plants can vary greatly (up to threefold) for the same variety by growth conditions, geographical areas, years, biotic stress factors (e.g., pests), and abiotic stress factors, such as temperature, nutritional status, or drought [4]. Dietary culture has an especially big influence on isoflavone content in the diet. Asian and vegetarian diets provide 20–50 mg isoflavones/day, in some cases reaching 100 mg/day, while the Western diet contributes only 0.2–1.5 mg isoflavones/day [2]. Based on recent report of European Food Safety Authority (EFSA), in Europe the dietary isoflavone intake is usually under 1 mg/day, despite an increase in the soy food consumption [17]. The differences between the types of diets refer to the amount of isoflavone in foods, as well as the type of food consumed. In the Western diet, solid processed soy products (such as tofu) and soymilk dominate the diet, and they contain both glycosides (genistin and daidzin which are stable during processing) and aglycones. In the Asian diet, most soy products are obtained through fermentation and have higher amounts of aglycones [3]. Miso, fermented soybean paste (Japan); doenjang, fermented soybean paste (Korea); douchi, fermented soybeans (China); and tempeh, fermented soybean cake (Indonesia) are staple foods in some Asian countries. Simultaneously, health benefit probiotics are formed in these foods during the fermentation processes [18].
Besides soy, other plants in the Fabaceae family have a high content of isoflavones: species of clover, mainly red clover (
2.3. Mechanism of estrogen-like action of isoflavones
According to the xenohormesis theory, plants synthesize phytochemicals to withstand and adapt under stress. Indeed, isoflavone biosynthesis depends on the environmental conditions in which the plant grows and is stimulated by stress. The stress-induced plant compounds have the ability to upregulate stress adaptive pathways in animals and humans. In the body, the biological effects of isoflavones are exercised by modulating pathways mediated by estrogen receptors (ERs) or various key enzymes involved in cellular signaling or metabolism and antioxidant potential [4].
3. The estrogenic/antiestrogenic effects
Isoflavones produce both estrogenic and antiestrogenic effects through several ways. Due to their structure similar to that of 17
Isoflavones induce agonist/antagonist effects depending on the level of the endogenous estrogen. For people with high levels of estrogen, (women premenopause, especially in the follicular phase of the menstrual cycle), the isoflavones bind to the estrogen receptors. Because of their weak estrogen potency, isoflavones exert an antagonist effect. They block the action of endogenous estrogens on their receptors. In case of low concentration of endogenous estrogens (women in menopause, after ovariectomy, or males), the estrogenic action of isoflavones becomes evident, showing additive agonist effect [34]. This is the reason why isoflavones can be used as a long-term complementary or alternative hormone therapy [35].
Isoflavones and their active metabolites can bind to the membrane ERs and induce rapid non-genomic effects by which they modulate cellular metabolism. Thus, they can change the protein kinase and lipid kinase cell signaling pathways [1]. It is believed that the activation of these signaling pathways by isoflavones causes some beneficial effects, in particular in the tissues that are not specific targets for the estrogens. At the circulatory system, the isoflavones induce vasodilation by increasing the production of nitric oxide (NO) after the activation of the endothelial NO− synthase. At the central nervous system, they improve the cognitive function by affecting cell membrane permeability and altering the neuronal excitability. In the skeletal system, the isoflavones inhibit the tyrosine kinase causing changes in the alkaline phosphatase activity. On the other hand, they induce the apoptosis of the osteoclasts, suppress the formation of osteoclasts [34], and prevent the bone demineralization [35].
Also, isoflavones influence the activity of some of the enzymes involved in the metabolism of the sex steroid hormones. In this way they inhibit 5α-reductase (the enzyme responsible for the conversion of testosterone to 5α-dihydrotestosterone) and aromatase (involved in the conversion of testosterone to estradiol) in low concentrations, but they increase the aromatase activity at high concentrations. Isoflavones have an affinity for sex hormone-binding globulin (SHBG) and they induce its expression. Therefore, they affect the free-steroid hormone level in the systemic circulation. But these outcomes depend on many factors, including species, gender, and the hormonal status [35].
Xenoestrogens can modulate the enzyme activity of aromatase. Thus, they induce alterations in the metabolism of fats and carbohydrates through effects on ERα. The decrease of endogenous estrogen levels on ERα, aromatase inhibition or the existence of mutations affecting the enzyme activity has been correlated with visceral obesity or truncate, hyperlipidemia, glucose intolerance and insulin resistance, low physical activity, and reduced energy expenditure. Isoflavones compensate for the deficit of estrogens and have the ability to prevent the associated negative effects. Asian diets, rich in isoflavones, are correlated with low incidence of obesity and metabolic syndrome, favorable plasma profile, and a reduced body mass index in postmenopausal women [4].
4. Health benefits of isoflavones
4.1. Isoflavones and their effects on diseases
Numerous epidemiological and clinical studies have demonstrated the protective role of dietary isoflavones against development of specific menopause symptoms [36–38] and several chronic diseases, including cardiovascular diseases [39, 40], osteoporosis [38], cognitive impairment [37], and hormone-dependent cancers [41–43]. Based on human health benefits of soy diet, the Food and Drug Administration (FDA) approved the use of the following health claim on the labels: “25 grams of soy protein a day, as part of a low in saturated fat and cholesterol, may reduce the risk of heart disease” [44].
Isoflavones, as all polyphenols, have a strong antioxidant activity. They can neutralize free radicals and prevent the lipid peroxydation by stopping the chain reactions. Also, isoflavones induce the antioxidant enzymes (glutathione peroxidase, catalase, and superoxide dismutase) and inhibit the expression of some enzymes, such as xanthine oxidase [1]. The antioxidant protective action of isoflavones from soy or plant extracts, such as
4.2. Anticarcinogenic activity of isoflavones
The anticarcinogenic potential of isoflavones is based on multiple actions: binding to estrogen receptors (ERs), changing of cell signaling pathways, and inhibition of the key enzymes involved in the metabolism of sex hormones. Also, the anticarcinogenic potential of isoflavones has positive effects through independent mechanisms which do not involve ERs, such as antioxidant activity, reduction in the bioactivation of carcinogens, and stimulation of detoxification [2, 48].
Anticarcinogenic activity of genistein has been assessed more thoroughly among isoflavones. Genistein initiates apoptosis, alters cell proliferation and angiogenesis, and inhibits metastasis in many types of cancer cells [49]. It is a tyrosine kinase inhibitor. Therefore, in breast cancer cells, it slows down tumorigenesis; in the circulatory system, it prevents tumor vascularization; in the nervous system, it induces neuroprotective effects. In addition, genistein affects tumorigenesis by inhibiting DNA topoisomerases I and II [50], alteration of epigenetic regulations (both histone methylation and DNA methylation), and activating tumor suppressor genes [51]. As a polyphenol, genistein has antioxidant [1] and anti-inflammatory potential [52]. Another possible action pathway for genistein is the competitive inhibition of estrone metabolism through cytochrome P450 isoenzymes by altering the 2-hydroxy-estrone (2-OH-E1)/16α-hydroxy-estrone (16α-OH-E1) ratio, as noticed in vitro [53]. While 2-OH-E1 is a weak estrogen, 16α-OH-E1 has an important role in carcinogenesis, showing a strong estrogen effect and genotoxic properties [54]. 16α-OH-E1 covalently binds to the estrogenic receptors and thus stimulates cell proliferation [55]. The ratio 2-OH-E1/16α-OH-E1 has been proposed and studied as a biomarker of breast cancer risk [55–59], but now its significance is controversial. In high concentrations, genistein decreases the hydroxylation of estrone in position 2 in favor of hydroxylation in position 16α [55]. Other studies show that genistein has no mutagenic or clastogenic activity in vivo. But in high concentration of genistein, it has clastogenic potential in vitro, explained by the topoisomerase inhibitory effect, which is known to cause chromosome damage above a certain threshold dose [60].
Anti-proliferative effects of high concentrations of genistein were demonstrated in all breast cancer cells, both ER positive and ER negative. However, there are several studies showing that genistein shows both anti-proliferative and proliferative effects, depending on the concentration, type of tumor, level of endogenous estrogens present in the tissue, or development stage. At low physiological concentrations, genistein stimulates tumorigenesis and cancels the effects of tamoxifen in ER-positive breast cancer cells [50]. Similar dual effects were observed in the case of tamoxifen and other selective estrogen receptor modulators (SERMs) [16].
In fermented soybean products (e.g., natto, miso, tempeh), aglycons can suffer changes under the effect of enzymes produced by the microorganisms involved in the fermentation process. Thus,
Equol has a higher estrogenic potential than daidzein, its precursor, and a preferential affinity for ER
4.3. Effects of isoflavones on hormone-dependent cancers
Clinical studies show contradictory results of the efficacy of isoflavones in the treatment of breast cancer. The effects depend on a number of factors such as age, gender, hormonal status, type of isoflavones consumed (soy proteins or isolated isoflavones), dose, diet (type of food), and extent of consumption [2].
A recent meta-analysis of 35 studies shows that soy isoflavones lower the risk of breast cancer in both premenopausal and post-menopausal women. The effect is more evident in Asian women than in those living in Western countries, probably due to differences in quality (traditionally fermented foods) and quantity of the isoflavone products ingested [41]. In Asian women, a diet rich in soy food lowers breast cancer risk with 30% [61]. A higher prevalence of equol-producer phenotype in Asian population can be an essential factor. Equol-producer phenotype is associated with a substantial reduction in the risk of breast cancer. Several specific biomarkers are favorable modified, such as sex hormone-binding globulin (SHBG) and steroid hormone levels in plasma, a higher urinary 2-hydroxy-estrone/16α-hydroxy-estrone ratio, and a lower mammographic breast density [2]. However, because several studies have provided mixed or contradictory results, the general recommendation for patients diagnosed with estrogen-dependent breast cancer is to avoid consuming high quantities of products containing isoflavone. Indeed, isoflavones are selective estrogen receptor modulators (SERMs), and their effects would depend on multiple factors.
Another meta-analysis of five cohort studies that included more than 11,000 female patients diagnosed with breast cancer focused on the post-diagnostic relationship between consumption of soy foods and mortality or cancer recurrence. The study concluded that the ingestion of soy foods reduced mortality and recurrence in all types of breast cancer, especially in the ER-negative, ER-positive/PR-positive, and postmenopausal patients [42]. In women diagnosed with breast cancer under tamoxifen treatment, the consumption of plants containing isoflavones did not alter plasma levels of the drug and its metabolites [62]. Moreover, a recent study shows that a moderate intake of soy isoflavones (5–10 g soy protein/day) would have an optimal effect on tamoxifen treatment on these patients [63].
In some studies [64], excessive consumption of soy was associated with a negative impact on male fertility and reproductive hormones and the disruption of the thyroid gland function. In other studies these effects were inconsistent [65].
Isoflavones can modulate the toxicity of other xenoestrogens, but the interactions are complex and difficult to predict relying only on in vitro steroid receptor affinities [66]. In these kinds of interactions, multiple mechanisms are involved, both estrogen and non-estrogen type, such as oxidative stress [32, 47, 53]. European Food Safety Authority (EFSA) has recently conducted a systematic study of published medical literature, focusing on the correlation between the intake of soy isoflavones and the induced effects on the breast (mammographic density, proliferative marker Ki67 expression), uterus (endometrial thickness, histopathology changes), and thyroid (the thyroid hormone). Results showed that the intake of 35–150 mg isoflavones/day does not affect these organs in peri- and postmenopausal women [17]. Isoflavones have demonstrated prostate cancer efficacy in several studies: in vitro, on prostate cancer cell lines, in vivo, and in numerous clinical trials [43, 67, 68]. Conclusion of a recent meta-analysis suggests that phytoestrogen intake, mostly genistein and daidzein, can be correlated with a decreased risk of prostate cancer [69].
5. Recent advances in analytical methods of isoflavones
5.1. Isolation of isoflavones in foods and vegetable materials
In recent years, due to the health benefits provided by isoflavones, higher attention has been paid to the analytical methods that allow identification and quantification of isoflavones from different types of samples: (a) food, for dietary intake assessing [15, 70]; (b) food supplements, for standardization of nutraceuticals [5, 71]; (c) vegetable products, for phytotherapeutic evaluation [19, 20, 28]; and (d) human biological samples (plasma, urine) [5]. These analytical methods are commonly used for isoflavone bioavailability assessing and in pharmacokinetic or pharmacological studies.
Isoflavones are solubilized from food or vegetable material by refluxing or maceration, shaking, and stirring [72]. The isolation of isoflavones from the mixture can be achieved either by conventional methods, liquid-liquid extraction [11, 15, 19] or Soxhlet, or by modern ones—supercritical fluid extraction, ultrasound-assisted extraction [19, 71], pressurized fluid extraction, microwave-assisted extraction, and solid-phase extraction [5, 73] (Table 4).
Analytes | Sample | Extraction method | Detection | Run time (min) | LOQ | References |
---|---|---|---|---|---|---|
3 IFs | Soy dry extract | Sonication/Steam bath | HPLC-DAD | 20 | 40–100 ng/mL | [71] |
3 IFs, Cou* | 10 plant species | UAE** | ULPC-PDA | 4 | 1.97–4.08 ng/mL | [19] |
12 IFs | Soybean seeds | Maceration | HPLC-UV | 60 | NA# | [70] |
12 IFs | Soybeans, soy products | Maceration | HPLC-DAD | 30 | <600 nmol/L | [72] |
3 IFs | Coffee | Refluxing | HPLC-DAD | 35 | 13.7–25.0 ng/mL | [14] |
17 IFs | Soymilk | Refluxing | LC-ESI(+)-MS/MS | NA# | NA# | [74] |
7 IFs, Cou* | 2 plant extracts | Refluxing or Maceration | LC-ESI(−)-MS/MS | 18 | 40 ng/mL | [20] |
5 IFs, Cou* | 7 plant extracts | Maceration or percolation | ULPC-ESI(+)-MS/MS | 5.5 | 5–10.78 ng/mL | [28] |
5 IFs | Legumes | SPE C18 | UHLPC-ESI(+)-MS/MS | 18 | 0.1–1 ng/mL | [73] |
5 IFs | Coffee | SPE C18 | HPLC-ESI-MS/MS | 18 | 0.05–1 ng/mL | [75] |
The methods used to isolate isoflavones from food are selected function of the nature of the food, the type of the isoflavones analyzed (the total of aglycones or aglycones and glycosides), and the instrumental method used for identification and quantification. Several examples are presented below.
Liggins et al. isolated isoflavones from cereals and derivatives after a prior sonication in a polar solvent (methanol/water 4:1, v/v), in order to break apart the cellular material, followed by filtration and evaporation of the solvent under nitrogen. In order to determine the total aglycones, glycosides were hydrolyzed in an acid medium (0.1 M acetate buffer, pH 5) by overnight incubation at 37 °C in the presence of cellulase (enzyme used for hydrolytic removal of the hydrolysis resulted carbohydrates). Aglycones were extracted into ethyl acetate and were derivatized and analyzed using GC-MS [15]. Otieno et al. analyzed isoflavones from fermented and unfermented soy milk. For the solubilization of analytes, the freeze-dried sample was refluxed in methanol for 1 hour and filtered, and after adding the internal standard, the solvent has been evaporated to dryness under nitrogen. The residue has been suspended into a buffer (10 mm ammonium acetate containing 0.1% trifluoroacetic acid) and centrifuged, and the supernatant was filtered and analyzed using high-performance liquid chromatography (HPLC) [74].
Extraction and analysis of isoflavones in soybeans can be realized through maceration of the powdered beans with 70% ethanol at room temperature, for 24 hours under constant stirring. After centrifugation and filtering, the supernatant is analyzed directly by HPLC [70]. Also, analysis of isoflavones contained in food supplements requires a simple preparation of the samples: fine powdering of tablets, refluxing in 80% methanol for 1 hour, filtering, and injection into the HPLC system [5].
Hydroalcoholic extracts or tinctures can be prepared from either fresh or dry and pulverized vegetable materials. The hydroalcoholic extracts can be made in 70% ethanol or methanol, by refluxing and filtration; by cold maceration, pressing, and filtration [20]; by percolation [28]; or using modern methods, such as ultrasound-assisted extraction in 50% ethanol [19]. The extracts can be analyzed directly by LC-MS/MS, after an adequate dilution [20], or they can be subjected to an acid hydrolysis [19] in order to release aglycones. Further, the aglycones can be assessed directly or after liquid-liquid extraction, for a concentration of the analytes [19].
In biological samples (e.g., plasma and human urine) isoflavones can be found in different forms: as aglycones (active metabolites), aglycone derivatives (with or without bioactivity), or conjugated metabolites (
5.2. Quantification of isoflavones in foods and vegetable materials
For isoflavone identification, the following chromatographic methods are used: gas chromatography coupled with mass spectrometry (GC-MS) [5, 15], high-performance liquid chromatography (HPLC) with UV detector (photodiode array, PDA) [28, 70, 71], fluorescence detector (FLD), electrochemical detector (ECD) or mass spectrometer detector (MS) [20, 74, 75], and, less often, capillary electrophoresis (CE).
Quantification of isoflavones and their derivatives can be achieved in two ways: (a) by determining the free aglycons after a prior acid hydrolysis [19, 70, 72], alkaline hydrolysis [72], or enzymatic hydrolysis [72] of the glycosides in the sample and (b) by simultaneously analyzing the glycosides and aglycones present in the sample [20, 28]. GC-MS methods are used less lately, because they require an additional step of isoflavone derivatization to the volatile compounds [5, 15]. This additional step increases both the time and the cost of the analysis and represents a potential source of error [28].
Generally, HPLC-UV is not sensitive enough (Table 4) for the quantification of small levels of isoflavones from plant extracts [19] or human plasma [5]. This method often requires a hydrolysis step to transform glycosides into aglycones followed by the quantification of total aglycones from the sample [71].
In order to correctly identify new isoflavones or isoflavone derivatives present in the samples analyzed, liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) are the preferred methods (Table 4), due to the advantages: speed, selectivity, sensitivity, and robustness. In addition, mass spectrometry detection allows sure determination of the compounds based on molecular weight and ion charge. For the quantification of isoflavones, the pseudo-molecular ions or the ionic fragments resulted after fragmentation are monitored. In LC-MS/MS analysis, compound identification can be achieved even if their separation is not complete, and it is an advantage [74]. A shorter analysis can be realized by ultra-performance liquid chromatography (UPLC) [19, 28]. This method uses columns with very small size of the packing particles (1.7 μm) and consequently performs separations with superior resolution in a shorter time and a lower consumption of the mobile phase.
The isoflavones have polyphenolic structure and can easily lose a proton to form negative pseudo-molecular ions [M-H]− [20]. However, they can also be detected after ionization in positive mode to [M + H]+ [74]. Isoflavones are polar compounds and they form ions in solution. For these type of compounds, electro-spray ionization (ESI) is the most commonly used source to obtain analytical ions. Atmospheric pressure chemical ionization (APCI) is the source preferred for non-polar analytes that ionize in the gas phase. The isoflavones often give poor response in this ionization source [28]. The fragmentation patterns of isoflavone glycosides (malonyl-glycosides, acetyl-glycosides, glycosides, aglycones) follow a similar trend. However each compound has a unique fragmentation pattern that allows their accurate identification (Table 5) [74].
Isoflavone | [M + H]+ | Transitions | [M − H]− | Tranzitions |
---|---|---|---|---|
Daidzein | 255 [28, 74] | →199 [28] | 253 [20, 73, 75] | →208, 132 [73, 75] |
Formononetin | 269 [28] | →197 [28] | 267 [20, 75] | →252, 223 [75] |
Genistein | 271 [28, 73, 74] | →153 [28] | 269 [20, 73, 75] | →159, 133 [73, 75] |
Biochanin A | 283 [73, 75] | →268, 239 [73, 75] | ||
Glycitein | 285 [74] | →270, 257, 229, 196, 166 [74] | 283 [20] | |
Daidzin | 417 [28, 73, 74] | →255 [28, 73, 74], 199 [73] | 415 [20] | →253 [20] |
Ononin | 429 [20] | →267 [20] | ||
Genistin | 433 [28, 74, 75] | →271 [28, 73–75], 91 [73, 75] | 431 [20] | →268, 269 [20] |
Glycitin | 447 [74] | →428, 285 [74] | ||
Ac-daidzin | 459 [74] | →441, 255 [74] | ||
Ac-genistin | 475 [74] | →431, 271 [74] | ||
Ac-glycitin | 489 [74] | →471, 285 [74] | ||
Mal-daidzin | 503 [74] | →485, 255 [74] | ||
Mal-genistin | 519 [74] | →501, 271 [74] |
6. Conclusion
Dietary intake of isoflavones is widespread, mainly due to the high consumption of soybean products. Health benefits of isoflavones justify the interest for this class of bioactive compounds, but the controversial outcomes of some clinical and epidemiological studies require further investigations. In the context of these researches, the analytical methods applied for assessment of isoflavones are very valuable. They allow for the evaluation of dietary intake of isoflavones, equating the health benefits and the circumstances in which they are exerted, and highlight the natural sources of isoflavones with phytotherapeutic potential.
References
- 1.
Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients. 2010;2(12):1231–1246. Doi: 10.3390/nu2121231 - 2.
Bolca S. Bioavailability of soy-derived isoflavones and human breast cancer. In: Watson RR, Preedy VR, Zibadi S, editors. Polyphenols in Human Health and Disease. Amsterdam: Elsevier; 2014. pp. 1241–1256. Doi: 10.1016/B978-0-12-398456-2.00094-3 - 3.
Setchell KD, Brown NM, Zimmer-Nechemias L, Brashear WT, Wolfe BE, Kirschner AS, Heubi JE. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. Am J Clin Nutr. 2002;76(2):447–453. - 4.
Cederroth CR, Nef S. Soy, phytoestrogens and metabolism: a review. Mol Cell Endocrinol. 2009;304(1–2):30–42. Doi: 10.1016/j.mce.2009.02.027 - 5.
Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr. 2001;131(4 Suppl):1362S–1375S. - 6.
Tolleson WH, Doerge DR, Churchwell MI, Marques MM, Roberts DW. Metabolism of biochanin A and formononetin by human liver microsomes in vitro . J Agric Food Chem. 2002;50(17):4783–4790. - 7.
Gaya P, Medina M, Sánchez-Jiménez A, Landete JM. Phytoestrogen metabolism by adult human gut microbiota. Molecules. 2016;21(8),1034:1-17. Doi: 10.3390/molecules21081034 - 8.
Setchell KD, Clerici C. Equol: history, chemistry, and formation. J Nutr. 2010;140(7):1355S–1362S. Doi: 10.3945/jn.109.119776 - 9.
Douglas CC, Johnson SA, Arjmandi BH. Soy and its isoflavones: the truth behind the science in breast cancer. Anticancer Agents Med Chem. 2013;13(8):1178–1187. Doi: 10.2174/18715206113139990320 - 10.
Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein contents of vegetables. Br J Nutr. 2000;84(5):717–725. - 11.
Megías C, Cortés-Giraldo I, Alaiz M, Vioque J, Girón-Calle J. Isoflavones in chickpea (Cicer arietinum) protein concentrates. J Funct Foods. 2016;21:186–192. Doi: 10.1016/j.jff.2015.12.012 - 12.
USDA Database for the Isoflavone Content of Selected Foods Release 2.1. 2015. https://www.ars.usda.gov/ARSUserFiles/80400525/Data/isoflav/Isoflav_R2-1.pdf. - 13.
Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein content of fruits and nuts. J Nutr Biochem. 2000;11(6):326–331. - 14.
Alves RC, Almeida IMC, Casal S, Oliveira MBPP. Method development and validation for isoflavones quantification in coffee. Food Chem. 2010;122:914–919. Doi: 10.1016/j.foodchem.2010.03.061 - 15.
Liggins J, Mulligan A, Runswick S, Bingham SA. Daidzein and genistein content of cereals. Eur J Clin Nutr. 2002;56(10):961–966. - 16.
Patisaul HB, Jefferson W. The pros and cons of phytoestrogens. Front Neuroendocrinol. 2010;31(4):400–419. Doi: 10.1016/j.yfrne.2010.03.003 - 17.
EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food). Scientific opinion on the risk assessment for peri- and post-menopausal women taking food supplements containing isolated isoflavones. EFSA J 2015;13(10):4246. Doi: 10.2903/j.efsa.2015.4246 - 18.
Chang TS. Isolation, bioactivity, and production of ortho-hydroxydaidzein and ortho-hydroxygenistein. Int J Mol Sci. 2014 Apr 3;15(4):5699–5716. Doi: 10.3390/ijms15045699 - 19.
Kiss B, Popa DS, Hanganu D, Pop A, Loghin F. Ultra-performance liquid chromatography method for the quantification of some phytoestrogens in plant material. Rev Roum Chim. 2010;55(8):459–465. - 20.
Vlase L, Popa DS, Tero-Vescan A, Olah N. New liquid chromatography: mass spectrometry assay for natural phytoestrogens from vegetable extracts. Acta Chromatogr. 2011;23:509–520. - 21.
Hanganu D, Vlase L, Olah N. LC/MS analysis of isoflavones from fabaceae species extracts. Farmacia, 2010;58(2):177–183. - 22.
Valls J, Millán S, Martí MP, Borràs E, Arola L. Advanced separation methods of food anthocyanins, isoflavones and flavanols. J Chromatogr A. 2009;1216(43):7143–7172. Doi: 10.1016/j.chroma.2009.07.030 - 23.
Kicel A, Wolbiś M. Phenolic content and DPPH radical scavenging activity of the flowers and leaves of Trifolium repens. Nat Prod Commun. 2013;8(1):99–102. - 24.
Mu H, Bai YH, Wang ST, Zhu ZM, Zhang YW. Research on antioxidant effects and estrogenic effect of formononetin from Trifolium pratense (red clover). Phytomedicine. 2009 Apr;16(4):314–319. Doi: 10.1016/j.phymed.2008.07.005 - 25.
Beck V, Rohr U, Jungbauer A. Phytoestrogens derived from red clover: an alternative to estrogen replacement therapy? J Steroid Biochem Mol Biol. 2005;94(5):499–518. Doi: 10.1016/j.jsbmb.2004.12.038 - 26.
Grigorescu E, Ciulei I, Stanescu U. Index fitoterapeutic. Bucuresti: Editura Medicala; 1986. p. 252. - 27.
Bora KS, Sharma A. Phytochemical and pharmacological potential of Medicago sativa: a review. Pharm Biol. 2011;49(2):211–220. Doi: 10.3109/13880209.2010.504732 - 28.
Kiss B, Popa DS, Păltinean R, Loghin F. A high-throughput UPLC-MS/MS for the simultaneous analysis of six phytoestrogens from Genista tinctoria extracts. J Liq Chromatogr Relat Technol. 2012;35(19):2735–2752. - 29.
Tero-Vescan A, Imre S, Vari CE, Osan A, Dogaru MT, Csedo C. Determination of some isoflavonoids and flavonoids from Genista tinctoria L. by HPLC-UV. Farmacia, 2009;57(1):120–127. - 30.
Rauter AP, Martins A, Lopes R, Ferreira J, Serralheiro LM, Araújo ME, Borges C, Justino J, Silva FV, Goulart M, Thomas-Oates J, Rodrigues JA, Edwards E, Noronha JP, Pinto R, Mota-Filipe H. Bioactivity studies and chemical profile of the antidiabetic plant Genista tenera. J Ethnopharmacol. 2009;122(2):384–393. Doi: 10.1016/j.jep.2008.10.011 - 31.
Rigano D, Cardile V, Formisano C, Maldini MT, Piacente S, Bevelacqua Y, Russo A, Senatore F. Genista sessilifolia DC. and Genista tinctoria L. inhibit UV light and nitric oxide-induced DNA damage and human melanoma cell growth. Chem Biol Interact. 2009;180(2):211–219. Doi: 10.1016/j.cbi.2009.02.010 - 32.
Popa DS, Bolfă P, Kiss B, Vlase L, Păltinean R, Pop A, Cătoi C, Crişan G, Loghin F. Influence of Genista Tinctoria L. or methylparaben on subchronic toxicity of bisphenol A in rats. Biomed Environ Sci. 2014;27(2):85–96. Doi: 10.3967/bes2014.021 - 33.
Hanganu D, Olah NK, Benedec D, Mocan A, Crisan G, Vlase L, Popica I, Oniga I. Comparative polyphenolic content and antioxidant activities of Genista tinctoria L. and Genistella sagittalis (L.) Gams (Fabaceae). Pak J Pharm Sci. 2016;29(1 Suppl):301–307. - 34.
Hwang CS, Kwak HS, Lim HJ, Lee SH, Kang YS, Choe TB, Hur HG, Han KO. Isoflavone metabolites and their in vitro dual functions: they can act as an estrogenic agonist or antagonist depending on the estrogen concentration. J Steroid Biochem Mol Biol. 2006;101(4–5):246–253. Doi: 10.1016/j.jsbmb.2006.06.020 - 35.
Pilšáková L, Riečanský I, Jagla F. The physiological actions of isoflavone phytoestrogens. Physiol Res. 2010;59(5):651–664. - 36.
Franco OH, Chowdhury R, Troup J, Voortman T, Kunutsor S, Kavousi M, Oliver-Williams C, Muka T. Use of plant-based therapies and menopausal symptoms: a systematic review and meta-analysis. JAMA. 2016;315(23):2554–2563. Doi: 10.1001/jama.2016.8012 - 37.
Cheng PF, Chen JJ, Zhou XY, Ren YF, Huang W, Zhou JJ, Xie P. Do soy isoflavones improve cognitive function in postmenopausal women?. Meta Anal Menopause. 2015;22(2):198–206. Doi: 10.1097/GME.0000000000000290 - 38.
Pawlowski JW, Martin BR, McCabe GP, McCabe L, Jackson GS, Peacock M, Barnes S, Weaver CM. Impact of equol-producing capacity and soy-isoflavone profiles of supplements on bone calcium retention inpostmenopausal women: a randomized crossover trial. Am J Clin Nutr. 2015;102(3):695–703. Doi: 10.3945/ajcn.114.093906 - 39.
Cavallini DC, Manzoni MS, Bedani R, Roselino MN, Celiberto LS, Vendramini RC, de Valdez G, Abdalla DS, Pinto RA, Rosetto D, Valentini SR, Rossi EA. Probiotic soy product supplemented with isoflavones improves the lipid profile of moderately hypercholesterolemic men: a randomized controlled trial. Nutrients. 2016;8(1),52:1–18. Doi: 10.3390/nu8010052 - 40.
Wong JM, Kendall CW, Marchie A, Liu Z, Vidgen E, Holmes C, Jackson CJ, Josse RG, Pencharz PB, Rao AV, Vuksan V, Singer W, Jenkins DJ. Equol status and blood lipid profile in hyperlipidemia after consumption of diets containing soy foods. Am J Clin Nutr. 2012;95(3):564–571. Doi: 10.3945/ajcn.111.017418 - 41.
Chen M, Rao Y, Zheng Y, Wei S, Li Y, Guo T, Yin P. Association between soy isoflavone intake and breast cancer risk for pre- and post-menopausal women: a meta-analysis of epidemiological studies. PLoS One. 2014;9(2):e89288. Doi: 10.1371/journal.pone.0089288 - 42.
Chi F, Wu R, Zeng YC, Xing R, Liu Y, Xu ZG. Post-diagnosis soy food intake and breast cancer survival: a meta-analysis of cohort studies. Asian Pac J Cancer Prev. 2013;14(4):2407–2412. Doi: http://dx.doi.org/10.7314/APJCP.2013.14.4.2407. - 43.
Zhang Q, Feng H, Qluwakemi B, Wang J, Yao S, Cheng G, Xu H, Qiu H, Zhu L, Yuan M. Phytoestrogens and risk of prostate cancer: an updated meta-analysis of epidemiologic studies. Int J Food Sci Nutr. 2016;9:1–15. Doi: 10.1080/09637486.2016.1216525 - 44.
US Food and Drug Administration. Guidance for Industry: A Food Labeling Guide (11. Appendix C: Health Claims). 2013. http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/LabelingNutrition/ucm064919.htm. - 45.
Azadbakht L, Kimiagar M, Mehrabi Y, Esmaillzadeh A, Hu FB, Willett WC. Dietary soya intake alters plasma antioxidant status and lipid peroxidation in postmenopausal women with the metabolic syndrome. Br J Nutr. 2007;98(4):807–813. Doi: 10.1017/S0007114507746871 - 46.
Cha YS, Park Y, Lee M, Chae SW, Park K, Kim Y, Lee HS. Doenjang, a Korean fermented soy food, exerts antiobesity and antioxidative activities in overweight subjects with the PPAR-γ2 C1431T polymorphism: 12-week, double-blind randomized clinical trial. J Med Food. 2014;17(1):119–127. Doi: 10.1089/jmf.2013.2877 - 47.
Popa DS, Hanganu D, Vlase L, Kiss B, Loghin F, Crişan G. Protective effect of Trifolium pratense extract on oxidative stress induced by bisphenol A in rats. Farmacia. 2014:62(2):341–349. - 48.
Lockwood B. Nutraceuticals. A Guide for Healthcare Professionals. Second edition, London: Pharmaceutical Press; 2007. pp. 192–200. - 49.
Spagnuolo C, Russo GL, Orhan IE, Habtemariam S, Daglia M, Sureda A, Nabavi SF, Devi KP, Loizzo MR, Tundis R, Nabavi SM. Genistein and cancer: current status, challenges, and future directions. Adv Nutr. 2015;6(4):408–419. Doi: 10.3945/an.114.008052 - 50.
Kwon Y. Effect of soy isoflavones on the growth of human breast tumors: findings from preclinical studies. Food Sci Nutr. 2014;2(6):613–622. Doi: 10.1002/fsn3.142. - 51.
Casati L, Sendra R, Sibilia V, Celotti F. Endocrine disrupters: the new players able to affect the epigenome. Front Cell Dev Biol. 2015;3:37. Doi: 10.3389/fcell.2015.00037 - 52.
Setchell KD, Clerici C. Equol: pharmacokinetics and biological actions. J Nutr. 2010;140(7):1363S–1368S. Doi: 10.3945/jn.109.119784 - 53.
Gheldiu AM, Popa DS, Loghin F, Vlase L. Oxidative metabolism of estrone modified by genistein and bisphenol A in rat liver microsomes. Biomed Environ Sci. 2015;28(11):834–838. Doi: 10.3967/bes2015.116 - 54.
Lee AJ, Mills LH, Kosh JW, Conney AH, Zhu BT. NADPH-dependent metabolism of estrone by human liver microsomes. J Pharmacol Exp Ther. 2002;300(3):838–849. - 55.
Eliassen AH, Missmer SA, Tworoger SS, Hankinson SE. Circulating 2-hydroxy- and 16alpha-hydroxy estrone levels and risk of breast cancer among postmenopausal women. Cancer Epidemiol Biomarkers Prev. 2008;17(8):2029–2035. Doi: 10.1158/1055-9965.EPI-08-0262 - 56.
Ziegler RG, Fuhrman BJ, Moore SC, Matthews CE. Epidemiologic studies of estrogen metabolism and breast cancer. Steroids. 2015;99(Pt A):67-75. Doi: 10.1016/j.steroids.2015.02.015 - 57.
Morimoto Y, Conroy SM, Pagano IS, Isaki M, Franke AA, Nordt FJ, Maskarinec G. Urinary estrogen metabolites during a randomized soy trial. Nutr Cancer. 2012;64(2):307–314. Doi: 10.1080/01635581.2012.648819 - 58.
Atkinson C, Skor HE, Dawn Fitzgibbons E, Scholes D, Chen C, Wähälä K, Schwartz SM, Lampe JW. Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to middle-aged women. J Steroid Biochem Mol Biol. 2003;86(1):71–77. - 59.
Lu LJ, Cree M, Josyula S, Nagamani M, Grady JJ, Anderson KE. Increased urinary excretion of 2-hydroxyestrone but not 16alpha-hydroxyestrone in premenopausal women during a soya diet containing isoflavones. Cancer Res. 2000;60(5):1299–1305. - 60.
McClain MR, Wolz E, Davidovich A, Bausch J. Genetic toxicity studies with genistein. Food Chem Toxicol. 2006;44(1):42–55. - 61.
Messina M. Impact of soy foods on the development of breast cancer and the prognosis of breast cancer patients. Forsch Komplementmed. 2016;23(2):75–80. Doi: 10.1159/000444735 - 62.
Hu YC, Wu CT, Lai JN, Tsai YT. Detection of a negative correlation between prescription of Chinese herbal products containing coumestrol, genistein or daidzein and risk of subsequent endometrial cancer among tamoxifen-treated female breast cancer survivors in Taiwan between 1998 and 2008: a population-based study. J Ethnopharmacol. 2015;169:356–362. Doi: 10.1016/j.jep.2015.04.028 - 63.
Braakhuis AJ, Campion P, Bishop KS. Reducing breast cancer recurrence: the role of dietary polyphenolics. Nutrients. 2016;8(9),547:1-15. Doi: 10.3390/nu8090547 - 64.
Chavarro JE, Toth TL, Sadio SM, Hauser R. Soy food and isoflavone intake in relation to semen quality parameters among men from an infertility clinic. Hum Reprod. 2008;23(11):2584–2590. Doi: 10.1093/humrep/den243 - 65.
D’Adamo CR, Sahin A. Soy foods and supplementation: a review of commonly perceived health benefits and risks. Altern Ther Health Med. 2014;20(Suppl 1):39–51. - 66.
You L, Sar M, Bartolucci EJ, McIntyre BS, Sriperumbudur R. Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor. Toxicol Sci. 2002;66(2):216–225. Doi: 10.1093/toxsci/66.2.216 - 67.
Hwang YW, Kim SY, Jee SH, Kim YN, Nam CM. Soy food consumption and risk of prostate cancer: a meta-analysis of observational studies. Nutr Cancer. 2009;61(5):598–606. Doi: 10.1080/01635580902825639 - 68.
van Die MD, Bone KM, Williams SG, Pirotta MV. Soy and soy isoflavones in prostate cancer: a systematic review and meta-analysis of randomized controlled trials. BJU Int. 2014;113(5b):E119–E130. Doi: 10.1111/bju.12435 - 69.
He J, Wang S, Zhou M, Yu W, Zhang Y, He X. Phytoestrogens and risk of prostate cancer: a meta-analysis of observational studies. World J Surg Oncol 2015;13:231. Doi: 10.1186/s12957-015-0648-9 - 70.
Sun J, Sun B, Han F, Yan S, Yang H, Kikuchi A. Rapid HPLC method for determination of 12 isoflavone components in soybean seeds. Agric Sci China. 2011;10(1):70–77. Doi: 10.1016/S1671-2927(09)60291-1 - 71.
César Ida C, Braga FC, Soares CD, Nunan Ede A, Pianetti GA, Condessa FA, Barbosa TA, Campos LM. Development and validation of a RP-HPLC method for quantification of isoflavone aglycones in hydrolyzed soydry extracts. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;836(1–2):74–78. Doi: 10.1016/j.jchromb.2006.03.030 - 72.
Shao S, Duncan AM, Yang R, Marcone MF, Rajcan I, Tsao R. Systematic evaluation of pre-HPLC sample processing methods on total and individual isoflavones in soybeans and soy products. Food Res Int. 2011;44:2425–2434. Doi: 10.1016/j.foodres.2010.12.041 - 73.
Vila-Donat P, Caprioli G, Maggi F, Ricciutelli M, Torregiani E, Vittori S, Sagratini G. Effective clean-up and ultra high-performance liquid chromatography–tandem mass spectrometry for isoflavone determination in legumes. Food Chem. 2015;174:487–494. Doi: 10.1016/j.foodchem.2014.11.047 - 74.
Otieno DO, Rose H, Shah NP. Profiling and quantification of isoflavones in soymilk from soy protein isolate using extracted ion chromatography and positive ion fragmentation techniques. Food Chem. 2007;105:1642–1651. Doi: 10.1016/j.foodchem.2007.04.036 - 75.
Caprioli G, Navarini L, Cortese M, Ricciutelli M, Torregiani E, Vittori S, Sagratini G. Quantification of isoflavones in coffee by using solid phase extraction (SPE) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). J Mass Spectrom. 2016;51(9):698–703. Doi: 10.1002/jms.3802