Sulforaphane, Epithiospecifier Proteins (ESP) or just Sulforadex for Autism

  

One reader of the last post on Sulforaphane raised the issue of whether she should cook her broccoli sprouts, to optimize her autism therapy.

This seemed a bit strange, since even the researchers at Johns Hopkins are eating their sprouts raw.  She does have a valid point.  It seems that while sprouts have large amounts of glucoraphanin and the required enzyme myrosinase, they also have something called Epithiospecifier Protein (ESP).  If there is much ESP present, instead of Sulforaphane you get a very similar compound called Sulforaphane Nitrile.  You can see that the “S” has been replaced by an “N”.

All is not lost, for those of you with sprouts growing in the kitchen.

Further research showed that the concentration of ESP in the sprouts peaks on the second day and that by day 5 has dropped dramatically.

It was also showed that raising the temperature of the sprouts to 60 degrees Celsius deactivated the ESP.  Heating Broccoli florets much beyond this then reduced the Sulforaphane produced, but not heating the sprouts.

Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli.

Abstract

Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 degrees C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 degrees C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.

So it would seem that if you want to eat the sprouts raw, you need to wait for five days before consuming them.  Not good to eat them when two days old.

If you cook them, you do risk affecting the myrosinase and then you might need to add back some more from another source, just as Nicole mentioned in her comment.  But some research implies the sprouts are heat stable.

This all starts to get rather complicated.

Personally I decided to buy freeze dried broccoli sprout powder from Australia.  They claim to measure for ESP, and there is very little.  Their myrosinase has not been deactivated in processing.

If true, their product is near ideal.  Is say near ideal, because one spoonful also has the taste of a plateful of broccoli.

Mine has now arrived and so I will serve one level teaspoonful a day.

Other research actually suggested that Daikon radish may be event better than broccoli.  Johns Hopkins chose to patent the broccoli.  In their research compound, they reacted broccoli sprouts with daikon radish sprouts to make a standardized Sulforaphane which is then freeze dried and kept frozen.

RADISH SPROUTS VERSUS BROCCOLI SPROUTS: A COMPARISON OF ANTI-CANCER POTENTIAL BASED ON GLUCOSINOLATE BREAKDOWN PRODUCTS

Comparison of bioactive phytochemical content and releaseof isothiocyanates in selected brassica sprouts

Daikon powder is readily available and is a potent source of heat stable myrosinase.

So I will seek to get the optimal output from my Australian sprout powder by adding a dash of Daikon powder.

A better way?  Sulforadex

This kitchen chemistry may all seem rather haphazard and indeed it is.

Rather than try and make 8 mg of Sulforaphane in your kitchen, would it not be better to buy 8 mg of standardized heat stable Sulforaphane in the pharmacy?

Sulforadex is potentially exactly that; it is an analog of Sulforaphane.  Trials have started in humans and at very much higher doses to check for toxicity and side effects.

Here is a link to the Phase 1 trial:-

http://www.richmondpharmacology.com/downloads/Publications/Evgen-BPS-poster-tiny-web.pdf

The only questions I have are:- is anyone 100% certain that Sulforaphane is the only beneficial compound produced by eating broccoli?  Is Sulforaphane the only compound present in Johns Hopkin’s frozen capsules?  When they react their broccoli sprouts with daikon sprouts in the lab, there are other compounds produced.

Monty, aged 11 with ASD, is by now remarkably accommodating when it comes to downing unappetizing potions.  NAC tastes pretty bad, unless you use the more expensive effervescent variety.  But this pales in comparison to what a spoonful of broccoli sprout powder tastes like (and looks like).

They also make this powder in capsule form, for those who can swallow them. 

The more appetising anti-oxidant would be a bar of high flavanol dark chocolate, as we discovered in the previous post.  As well as tasting better, it may quite possibly be just as effective.

Why not Cocoa Flavanols for Autism?

How to make Sulforaphane (Broccoli) at home

I hope he took his Sulforaphane

This month thousands of runners braved thick smog at the Beijing marathon, with some even donning masks as air pollution soared to 16 times the maximum recommended level.

Johns Hopkins have been trialing their Sulforaphane in China as a therapy to counter the health effects of air pollution.

It was proposed that the potent anti-oxidant and chemoprotective protective properties of Sulforaphane would be a cheap way to protect the health of people living in highly polluted environments.

Broccoli sprout beverage enhances detoxification of air pollutants in clinical trial

 or the actual study:-

Rapid and Sustainable Detoxication of Airborne Pollutants by Broccoli Sprout Beverage: Results of a Randomized Clinical Trial in China

Abstract

Broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can generate the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases (GST) and other cytoprotective enzymes. A broccoli sprout–derived beverage providing daily doses of 600 mmol glucoraphanin and 40 mmol sulforaphane was evaluated for magnitude and duration of pharmacodynamics action in a 12-week randomized clinical trial. Two hundred and ninety-one study participants were recruited from the rural He-He Township, Qidong, in the Yangtze River delta region of China, an area characterized by exposures to substantial levels of airborne pollutants. Exposure to air pollution has been associated with lung cancer and cardiopulmonary diseases. Urinary excretion of the mercapturic acids of the pollutants, benzene, acrolein, and crotonaldehyde, were measured before and during the intervention using liquid chromatography tandem mass spectrometry. Rapid and sustained, statistically

significant (P _ 0.01) increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde, were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive than in the null genotype, irrespective of study arm assignment.

Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks.

Now this blog is not about pollution, but you might be interested to know that such pollution not only increases cancer risk (plus respiratory diseases, of course) but also increases the incidence of autism.

  

Study Suggests Link Between Air Pollution Exposure And Autism

How to make Sulforaphane at Home

Hopefully you can now see the potential benefits of Sulforaphane.  As I said in the earlier post, twenty years has passed since Johns Hopkins discovered Sulforaphane and there have been numerous studies and experiments done.  What follows is just my synthesis and conclusions of that work.

1.     Eating Broccoli

Broccoli does contain glucosinolate and the required enzyme myrosinase.  If you eat copious amount of raw broccoli or very lightly cooked (steaming for 2 minutes) you will produce Sulforaphane in your body.  The amount required would be literally pounds/kilos each and every day, to come close the therapeutic doses.

Frozen broccoli has no active myrosinase and over-cooked broccoli has no myrosinase.

Clever tricks developed to get round this include:-

·        Eating a small piece of raw broccoli (to provide  myrosinase) with your cooked broccoli

 ·        Adding a tiny amount of daikon radish to frozen broccoli.  This is really a great idea, since only 0.25% Daikon is needed, you get 99.75% broccoli and will never even notice or taste the daikon.  The idea is that this should be done by the food processor when they freeze the broccoli, you would not do anything at home.

  

Modifying the processing and handling of frozen broccoli for increased sulforaphane formation.

Abstract

Frozen broccoli can provide a cheaper product, with a longer shelf life and less preparation time than fresh broccoli. We previously showed that several commercially available frozen broccoli products do not retain the ability to generate the cancer-preventative agent sulforaphane. We hypothesized that this was because the necessary hydrolyzing enzyme myrosinase was destroyed during blanching, as part of the processing that frozen broccoli undergoes. This study was carried out to determine a way to overcome loss of hydrolyzing activity. Industrial blanching usually aims to inactivate peroxidase, although lipoxygenase plays a greater role in product degradation during frozen storage of broccoli. Blanching at 86 °C or higher inactivated peroxidase, lipoxygenase, and myrosinase. Blanching at 76 °C inactivated 92% of lipoxygenase activity, whereas there was only an 18% loss in myrosinase-dependent sulforaphane formation. We considered that thawing frozen broccoli might disrupt membrane integrity, allowing myrosinase and glucoraphanin to come into contact. Thawing frozen broccoli for 9 h did not support sulforaphane formation unless an exogenous source of myrosinase was added. Thermal stability studies showed that broccoli root, as a source of myrosinase, was not more heat stable than broccoli floret. Daikon radish root supported some sulforaphane formation even when heated at 125 °C for 10 min, a time and temperature comparable to or greater than microwave cooking. Daikon radish (0.25%) added to frozen broccoli that was then allowed to thaw supported sulforaphane formation without any visual alteration to that of untreated broccoli.

2.     Eating Broccoli Sprouts

It was shown that broccoli seeds and broccoli sprouts (5 day old broccoli) contain highly concentrated amounts of glucosinolate and the required enzyme myrosinase.  It is reported to be about 20 times higher in sprouts than full grown broccoli.

Broccoli sprouts are eaten uncooked and so no myrosinase is lost in food preparation.

Following all the Johns Hopkins research and commercialization, in many parts of the world you can readily buy fresh broccoli sprouts, many sold by companies licensed by the company run by the son of the original researcher at John Hopkins.

It was reported that that original lead researcher tries to regularly eat 4oz (120g) a week of broccoli sprouts, which is not so much.

However if you want to achieve the therapeutic doses in the clinical trials this will not be enough.

Trials used between 50 and 150 micromoles of Sulforaphane.

Rather unhelpfully they do not equate this to a measure accessible to lay people. 

If you recall your high school chemistry just go to Wikipedia and look up Sulforaphane:

Molecular formula	C6H11NOS2
Molar mass	177.29 g/mol

To convert to grams you just multiply by 177.29.

So the trials used dosages between 8.8 mg and 26.6 mg of sulforaphane.

Most of these trials are in adults and most people reading this blog are interested in treating children, so let’s work with the figure of 8mg of sulforaphane.

Bioavailability and inter-conversion of sulforaphane and erucin in humansubjects consuming broccoli sprouts or broccoli supplement in a cross-over study design

Abstract

Broccoli consumption may reduce the risk of various cancers and many broccoli supplements are now available. The bioavailability and excretion of the mercapturic acid pathway metabolites isothiocyanates after human consumption of broccoli supplements has not been tested. Two important isothiocyanates from broccoli are sulforaphane and erucin. We employed a cross-over study design in which 12 subjects consumed 40 grams of fresh broccoli sprouts followed by a 1 month washout period and then the same 12 subjects consumed 6 pills of a broccoli supplement. As negative controls for isothiocyanate consumption four additional subjects consumed alfalfa sprouts during the first phase and placebo pills during the second. Blood and urine samples were collected for 48 hours during each phase and analyzed for sulforaphane and erucin metabolites using LC-MS/MS. The bioavailability of sulforaphane and erucin is dramatically lower when subjects consume broccoli supplements compared to fresh broccoli sprouts. The peaks in plasma concentrations and urinary excretion were also delayed when subjects consumed the broccoli supplement. GSTP1 polymorphisms did not affect the metabolism or excretion of sulforaphane or erucin. Sulforaphane and erucin are able to interconvert in vivo and this interconversion is consistent within each subject but variable between subjects. This study confirms that consumption of broccoli supplements devoid of myrosinase activity does not produce equivalent plasma concentrations of the bioactive isothiocyanate metabolites compared to broccoli sprouts. This has implications for people who consume the recommended serving size (1 pill) of a broccoli supplement and believe they are getting equivalent doses of isothiocyanates.

Following consumption of 40 g of alfalfa sprouts or 6 placebo pills, no SFN or ERN metabolites were detected in plasma or urine from the four subjects in the control group (Figure 1). In contrast subjects who consumed 40 g of broccoli sprouts (150 and 71 μmoles glucoraphanin and glucoerucin, respectively) or 6 supplement pills (121 and 40 μmoles glucoraphanin and glucoerucin, respectively) had considerable amounts of SFN and ERN metabolites in both plasma and urine.

In 12 hours about 145 micromols of SFN and ERN were excreted in urine.  From the chart it looks likes SFN:ERN is about 2:1.  So assume about 95 micromols of SFN (sulforaphane).

In the following study using frozen sulforaphane made at Johns Hopkins about 85 micromols were excreted in 12 hours 

Bioavailability of sulforaphane from two broccoli sprout beverages: Results of a short term, cross-overclinical trial in Qidong, China

In the Johns Hopkins trial above the dosage was 800 μmol of glucoraphanin in GRR (the blue lines above) and 150 μmol of sulforaphane in SFR beverages (green lines above). The drugs were mixed with mango juice and water.

We compare the green line with the earlier study and see that 40 g of sprouts is a similar dose to 150 μmol of Johns Hopkins sulforaphane.

Now I did ask Johns Hopkins how many grams of broccoli sprouts yields 50 μmol of Johns Hopkins sulforaphane.  They did reply and said that the level varies among sprouts and so it is impossible to say.

We have seen in this blog, to date, that while nothing is 100% certain in autism or autism therapies, once you have exceeded a certain level of probability, it is worth giving things a try.  If you wait for 100% certainty, you will never move.

So while you will never know exactly how much sulforaphane is in your sprouts, it does look like a fair estimate is 3.8 μmol /g.

So if you want 50 μmol, then you would need to eat about 13g of sprouts a day.

To achieve the adult dose of 150  μmol you would need to eat 40g of sprouts a day.

As a double check compare this to what the original lead researcher is reporting to be taking, for preventative therapy.  He takes 4 oz. a week.  This is 113.4g or 16g a day.

This dose appears not to have harmed him, and he is now 91 years old!

Paul Talalay

Paul Talalay was born in Germany , but emigrated to England with his family in 1933, shortly after the Nazi Party came to power. He was educated at Bedford School and, in 1940, he travelled to the United States to enter Massachusetts Institute of Technology where he majored in biology

Talalay's career has been devoted to cancer research and the achievement of early protection against cell damage. A pioneer in the field of chemoprotective research strategies, Talalay and his colleagues devised simple cell culture methods for detecting phytochemicals which appear to boost enzymes that detoxify carcinogens in the body. This work led to the isolation of sulforaphane, found in broccoli, as a potent inducer of detoxifying phase two enzymes. These findings, published in 1992,  attracted worldwide attention as a major breakthrough in understanding the potential link between cruciferous vegetable consumption and reduced cancer risk.

Since I have no signs of any other Germans appearing on my Dean’s List and there are already plenty of Americans, he goes down as a German.  Nikola Tesla had the same problem, with four countries claiming him as their own (USA, Austria, Croatia and Serbia).

 

3.     Mixing Daikin Powder with Broccoli Powder

Many people do not like eating broccoli.  I do suggest you try eating it raw; it really is not bad at all, and much better than the soggy, over cooked, variety.

For those preferring powders and pills, the third method involves mixing freeze dried Daikin Radish with freeze dried broccoli.

It turns out that while the myrosinase in broccoli is not heat or cold stable, the daikon radish root is a good source of heat stable myrosinase.  This radish is commonly used in Japan and is available cheaply in freeze dried form.

This is the powder that was proposed to be added to Frozen broccoli, so that it would be a source of sulforaphane.

Broccoli powder is produced in large volumes for the supplement industry, which package it in capsules and sell it on to you.

Why nobody thought of including active myrosinase from daikon radish is beyond me.  It is not expensive.

There appears to be one broccoli supplement that does actually do what it says on the label and produce some  sulforaphane.  Perhaps it includes some Daikin powder ?  It was tested in the US.

That supplement is made in Australia.  It is not cheap.

It is claimed that:-

A 1-gram serve of EnduraCell powder is equivalent to 12 grams of fresh sprouts (with their sulforaphane inhibitors deactivated) and contains 30mg of Glucoraphanin that yields 12 mg Sulforaphane.

Research has shown that generally broccoli supplements do not perform, perhaps this one is different?

4.     Combining Broccoli Sprouts with Broccoli Powder

Since broccoli sprouts, like daikin radish, contains copious amounts of myrosinase, you could also combine fresh broccoli sprouts with broccoli powder.  This has actually been studied in research projects and does work.

Sulforaphane absorption and excretion following ingestion of a semi-purified broccoli powder rich in glucoraphanin and broccoli sprouts in healthy men.

Abstract

Sulforaphane (SF) is a chemopreventive isothiocyanate (ITC) derived from the myrosinase-catalyzed hydrolysis of glucoraphanin, a thioglucoside present in broccoli. Broccoli supplements often contain glucoraphanin but lack myrosinase, putting in question their ability to provide dietary SF. This study compared the relative absorption of SF from air-dried broccoli sprouts rich in myrosinase and a glucoraphanin-rich broccoli powder lacking myrosinase, individually and in combination. Subjects (n=4) each consumed 4 meals consisting of dry cereal and yogurt with 2 g sprouts, 2 g powder, both, or neither. Blood and urine were analyzed for SF metabolites. The 24 h urinary SF recovery was 74%, 49%, and 19% of the dose ingested from broccoli sprouts, combination, and broccoli powder meals, respectively. Urinary and plasma ITC appearance was delayed from the broccoli powder compared to the sprouts and combination. A liver function panel indicated no toxicity from any treatment at 24 h. These data indicate a delayed appearance in plasma and urine of SF from the broccoli powder relative to SF from myrosinase-rich sprouts. Combining broccoli sprouts with the broccoli powder enhanced SF absorption from broccoli powder, offering the potential for development of foods that modify the health impact of broccoli products.

Conclusion

More good news is that when you make sulforaphane in the above fashion, you also make some other interesting substances; one of these is Indole-3-carbinol (I3C).  I3C itself has some extremely interesting properties for both cancer and autism.  I3C up-regulates a protein called PTEN, encoded by the PTEN gene.  PTEN is dysfunctional in autism and, in general terms, may need to be up-regulated.  Indole-3-carbinol is one of the few, safe, known, ways to up-regulate PTEN.  PTEN is also a tumor suppressor gene and so in people with some cancers, up-regulating PTEN will slow cancer progression.

Anyway, it really does look like broccoli may be good for cancer and autism.

Bon Appetit!