Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method

Abstract

(1) Background: There is increasing understanding of the potential health benefits of cruciferous vegetables. In particular sulforaphane (SFN), found in broccoli, and its metabolites sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC) have potent antioxidant effects that may offer therapeutic value. Clinical investigation of sulforaphane as a therapeutic antioxidant requires a sensitive and high throughput process for quantification of sulforaphane and metabolites; (2) Methods: We collected plasma samples from healthy human volunteers before and for eight hours after consumption of a commercial broccoli extract supplement rich in sulforaphane. A rapid and sensitive method for quantification of sulforaphane and its metabolites in human plasma using Liquid Chromatography–Mass Spectrometry (LC–MS) has been developed; (3) Results: The LC–MS analytical method was validated at concentrations ranging between 3.9 nM and 1000 nM for SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC and between 7.8 nM and 1000 nM in human plasma for SFN. The method displayed good accuracy (1.85%–14.8% bias) and reproducibility (below 9.53 %RSD) including low concentrations 3.9 nM and 7.8 nM. Four SFN metabolites quantitation was achieved using external standard calibration and in SFN quantitation, SFN-d8 internal standardization was used. The reported method can accurately quantify sulforaphane and its metabolites at low concentrations in plasma; (4) Conclusions: We have established a time- and cost-efficient method of measuring sulforaphane and its metabolites in human plasma suitable for high throughput application to clinical trials.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue

“…To account for potential adverse cardiac side effects induced by SFN during chronic treatment as it would be expected to occur in patients during longterm treatment, rat EHTs were continuously exposed to 1 μM SFN from the first day of casting for up to three weeks and NRCMs in 2D culture for 7 days. This concentration was previously reported in human plasma after SFN administration [ 17 , 18 ]. As observed before, the SFN-treated EHTs developed lower forces throughout the entire culture time (measurements took place between day 5 and day 22), when compared to vehicle (DMSO; 0.1%) or untreated controls, with no obvious changes in contraction kinetics (Supplemental Fig.…”

“…Due to the complexity of methods, subsequently, experiments were not performed for all SFN concentrations, but instead 30 μM was considered as an appropriate acute high bolus SFN concentration. As a chronic SFN intervention, exposure to 1 μM for the duration of 7 (2D) to three weeks (3D-EHTs) days in culture was used as this was within the concentration range of SFN plasma levels reported in human trials [ 17 , 18 ]. SFN exposure time for EHTs was increased compared to 2D cultures, to ensure full maturation and optimal contractile performance of the 3D heart muscle constructs.…”

“…SFN effects were investigated either acutely by short application of a spectrum of increasing SFN concentrations or where applicable one high SFN bolus concentration was applied. When administered chronically over the course of seven days, 1 μM was chosen as a low concentration that was within the range of detectable SFN plasma concentrations reported before in human trials [ 17 , 18 ]. SFN effects on EHT performance were investigated by analyzing spontaneous and electrically stimulated contraction patterns as a surrogate for unperturbed cardiomyocyte contractile function.…”

Eat Your Broccoli: Oxidative Stress, NRF2, and Sulforaphane in Chronic Kidney Disease

“…SFN also covalently modifies the N-terminal proline residue of macrophage migration inhibitory factor (MIF) [ 129 ] and inhibits its catalytic tautomerase activity [ 130 ]. SFN is also metabolized into several active metabolites: SNF–N-acetylcysteine (SFN-NAC), SFN-glutathione (SFN-GHS), and SFN-cysteine (SFN-Cys) [ 131 ]. Several of these metabolites have anticancer activities by modulating ERK1/2 phosphorylation [ 132 ] and microtubule function [ 132 , 133 ] although their biological actions have not been well studied.…”

The Challenges of Designing and Implementing Clinical Trials With Broccoli Sprouts… and Turning Evidence Into Public Health Action

“…This highly sensitive, simple, and convenient method continues to be used to assess the bioavailability of sulforaphane across a spectrum of formulations. In addition, methods have been developed to analyze the individual metabolites following their separation by liquid chromatography coupled with tandem mass spectrometry ( 66 , 83 – 85 ). Furthermore, the use of mass spectrometry coupled with stable isotope-labeled internal standards of sulforaphane [1-isothiocyanato-4-methyl-sulfinyl(1,1,2,2,3,3,4,4-2H8)butane] and its corresponding mercapturic acid pathway conjugates allows for quantitative, precise, sensitive, and specific analysis of sulforaphane and its metabolites in biospecimens ( 85 ).…”