Agaritine content in processed foods containing the cultivated mushroom (Agaricus bisporus) on the Nordic and the Czech market

The level of agaritine was measured in fresh and canned cultivated mushroom (Agaricus bisporus) as well as in other food products containing A. bisporus, by reversed phase high performance liquid chromatography. The two fresh samples were purchased on the open market and contained 212 and 229mg/kg, respectively. Of the 35 different trademarks of canned mushroom products studied, 25 were based on cut mushrooms and 10 on whole mushrooms. On average, whole mushrooms contained 14.9 ± 6.7 mg agaritine per kg product whereas cut mushrooms contained 18.1 ± 7.8mg mg/kg. There was no statistically significant difference between these two values. Agaritine levels in brine were generally slightly lower than the levels detected in canned mushrooms. Thus, the level of agaritine in A. bisporus is reduced more than 10 times during the wet canning process, resulting in low levels in canned products. On a portion basis, somewhat higher amounts of agaritine may be found in some other food products (mushroom soup and pasta sauce) containing A. bisporus.     

The content of agaritine in spores from Agaricus bisporus

Agaritine (l-glutamic acid, 5-(2-(4-(hydroxymethyl)phenyl)hydrazide)) was identified and quantified in spores of Agaricus bisporus by high resolution liquid chromatography with mass spectrometric detection using negative electrospray ionisation. The spores were collected from mushrooms purchased at the open marked in Oslo, and the agaritine was extracted in pure water before analyses. On average the agaritine content was 0.304 ± 0.003% of the spores.     

Determination of genotoxic phenylhydrazine agaritine in mushrooms using liquid chromatography-electrospray ionization tandem mass spectrometry

A new method with good sensitivity and specificity for detecting and quantifying genotoxic hydrazines, agaritine and 4-(hydroxymethyl)phenylhydrazine (HMPH), was developed using liquid chromatography-electrospray tandem mass spectrometry (MS). Synthetic agaritine and HMPH were structurally assigned by ¹H-, ¹³C- and two-dimensional nuclear magnetic resonance (NMR) analysis (HMBC and HMQC), high-resolution fast-atom bombardment (HR-FAB) MS and time of flight (TOF) MS. The polar molecule agaritine was separated on an ODS column using 0.01% AcOH-MeOH (99:1, v/v) as an eluent with a simple solid-phase extraction cleanup. There were no interference peaks for any of the mushrooms. Agaricus spp. contained 1247 and 2017 µg g^-1 agaritine. Other species of mushroom had no agaritine. Recoveries of agaritine from spiked mushroom samples were 60.3-114%. Intra-day precision values were 5.5 and 4.2%, and the inter-day precision values were acceptable (15.0 and 23.0%), as agaritine is unstable. The limit of quantification was 0.003 µg g^-1. Even a trace amount of agaritine in mushrooms can, therefore, be determined using this method. We also directly analysed HMPH, an active free hydrazine form of genotoxic agaritine, and obtained direct evidence of its absence from mushrooms. A precursor ion scan confirmed that agaritine derivatives, which could exert similar toxicity, were absent. The results indicate that this specific and sensitive analytical method for detecting and quantifying agaritine and its derivatives could help evaluate the risk of mushroom hydrazines to humans.     

Heat stability of agaritine in water extracts from Agaricus blazei and other edible fungi,and removal of agaritine by ethanol fractionation

An analytical system to quantify agaritine present in Agaricus blazei,Agaricus bisporus, and other food mushrooms was established using high pressure liquid chromatography combined with mass-spectroscopy (HPLC–MS). Water extracts from dried, homogenised mushrooms were kept at differing temperatures for defined periods to investigate the heat-stability of agaritine. Homogenates were then freeze-dried, and agaritine was extracted using methanol. After evaporation of methanol, agaritine levels were analysed by HPLC–MS. A. bisporus contained 341 ± 32 μg/g agaritine, and A. blazei contained 22–57 μg/g agaritine. While pure agaritine in H₂O solution was heat-unstable and decomposed exponentially at 120 °C, agaritine in Agaricus water extracts was partially heat stable, and 20–50% of agaritine remained after 120 min at 120 °C. Thus agaritine, a known carcinogen, is likely to be present in Agaricus extracts sold as nutritional supplements. Therefore, a method was developed that can be used to remove agaritine from water extracts in order to prevent health risk. Agaritine was successfully removed from Agaricus water extracts by ethanol fractionation.     

Stability of agaritine - a natural toxicant of Agaricus mushrooms

Agaritine (N-( n-^L( + )-glutamyl)-4-hydroxymethylphenylhydrazine) is a phenylhydrazine derivative found in the cultivated Agaricus mushroom which is claimed to give rise to carcinogenic products when metabolized. The stability of a synthetic sample of agaritine was tested in water and methanol. In tap water kept in open vials, agaritine was totally degraded within 48h. Since agaritine degradation was less pronounced in closed than in open vials, and slower in Milli Q water and, in particular, in Milli Q water purged with N₂, the degradation seems to be oxygen-dependent. The antioxidant dithiothreitol reduced the degradation. Four or possibly five ultraviolet-absorbing compounds were formed during degradation, but these have not yet been identified. Whereas the rate of degradation was similar at temperatures between 4 and 22°C, it was quicker at an acidic than at a neutral pH. The latter observation was confirmed in experiments where agaritine was incubated in simulated gastric fluid (pH 1.2). The importance of the degradation when performing toxicological studies with agaritine is discussed.     

Influence of storage and household processing on the agaritine content of the cultivated Agaricus mushroom

Agaritine (N-(γ-^L(+)-glutamyl)-4-hydroxymethyl-phenylhydrazine) was identified and quantified by high-pressure liquid chromatography and used as a marker for the occurrence of phenylhydrazine derivatives in the cultivated Agaricus bitorquis and A. garicus hortensis mushrooms. Although relatively high levels of agaritine (around 700 mg kg^-1) could be found in freshly harvested A. bitorquis from early flushes, samples from supermarkets contained less agaritine. The content of 28 samples varied between 165 and 457 mg kg^-1, on average being 272 ±69 mg kg^-1. The highest amounts of agaritine were found in the skin of the cap and in the gills, the lowest being in the stem. There was no significant difference in agaritine content of the two mushroom species in our study. Pronounced reduction in agaritine content was observed during storage of mushrooms in the refrigerator or freezer, as well as during drying of the mushrooms. The degree of reduction was dependent on the length and condition of storage and was usually in the region 20-75%. No reduction in agaritine content was observed during freeze-drying. Depending on the cooking procedure, household processing of cultivated Agaricus mushrooms reduced the agaritine content to various degrees. Boiling extracted around 50% of the agaritine content into the cooking broth within 5min and degraded 20-25% of the original agaritine content of the mushrooms. Prolonged boiling, as when preparing a sauce, reduced the content in the solid mushroom further (around 10% left after 2h). Dry baking of the cultivated mushroom, a process similar to pizza baking, reduced the agaritine content by approximately 25%, whereas frying in oil or butter or deep frying resulted in a more marked reduction (35-70%). Microwave processing of the cultivated mushrooms reduced the agaritine content to one-third of the original level. Thus, the exposure to agaritine was substantially less when consuming processed Agaricus mushrooms as compared with consuming the raw mushrooms. However, it is not yet known to what extent agaritine and other phenylhydrazine derivatives occurring in the cultivated mushroom are degraded into other biologically active compounds during the cooking procedure.