Occurrence of different trichothecenes and deoxynivalenol-3-ß-D-glucoside in naturally and artificially contaminated Danish cereal grains and whole maize plants.

Fusarium mycotoxins such as deoxynivalenol (DON) can occur in cereals conjugated to glucose and probably also to other sugars. These conjugates, which are often referred to as "masked mycotoxins", will not be detected with routine analytical techniques. Furthermore, it is suspected that the parent toxin may again be released after hydrolysis in the digestive tracts of animals and humans. Today, our knowledge of the occurrence of these compounds in cereal grains is limited. In this paper, a LC-MS/MS method for the simultaneous determination of DON, deoxynivalenol-3-ß-D-glucoside (DON-3-glucoside), 3 acetyl-DON, nivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin in naturally (n = 48) and artificially (n = 30) contaminated cereal grains (wheat, barley, oat, rye triticale) is reported. The method has also been applied to whole fresh maize plant intended for production of maize silage (n = 10). The samples were collected from the harvest years 2006-2010, The results show that DON-3-glucoside and DON co-occurred in cereal grains and, especially in several of the highly contaminated samples, the concentration of the glucoside can be relatively high, corresponding to over 37 % of the DON concentration. The DON-3-glucoside levels in both the naturally and in the artificially grain inoculated with Fusarium were second only to DON, and were generally higher than those of the other tested trichothecenes, which were found at low concentrations in most samples, in many cases even below the detection limit of the method. This argues for the importance of taking DON-3-glucoside into account in the ongoing discussion within the European Community concerning exposure re-evaluations for setting changed values for the tolerable intake for DON. Our results indicate that, in the naturally contaminated grains and in the Fusarium infested cereal grains (winter and spring wheat, oat, triticale), the concentration level of DON-3-glucoside is positively correlated to the DON content. When the DON concentration is high, then the content of DON-3-glucoside will most probably also be high and vice versa.

In vitro cytotoxicity of fungi spoiling maize silage.

Penicillium roqueforti, Penicillium paneum, Monascus ruber, Alternaria tenuissima, Fusarium graminearum, Fusarium avenaceum, Byssochlamys nivea and Aspergillus fumigatus have previously been identified as major fungal contaminants of Danish maize silage. In the present study their metabolite production and in vitro cytotoxicity have been determined for fungal agar and silage extracts. All 8 fungal species significantly affected Caco-2 cell viability in the resazurin assay, with large variations for each species and growth medium. The 50% inhibition concentrations (IC(50)) of the major P. roqueforti metabolites roquefortine C (48 µg/mL), andrastin A (>50 µg/mL), mycophenolic acid (>100 µg/mL) and 1-hydroxyeremophil-7(11),9(10)-dien-8-one (>280 µg/mL) were high. Fractionating of agar extracts identified PR-toxin as an important cytotoxic P. roqueforti metabolite, also detectable in maize silage. The strongly cytotoxic B. nivea and P. paneum agar extracts contained patulin above the IC(50) of 0.6 µg/mL, however inoculated onto maize silage B. nivea and P. paneum did not produce patulin (>371 µg/kg). Still B. nivea infected maize silage containing mycophenolic acid (∼50 mg/kg), byssochlamic acid and other metabolites, was cytotoxic. In contrast hot-spots of P. roqueforti, P. paneum, M. ruber and A. fumigatus were not more cytotoxic than uninoculated silage.

Multi-mycotoxin analysis of maize silage by LC-MS/MS.

This paper describes a method for determination of 27 mycotoxins and other secondary metabolites in maize silage. The method focuses on analytes which are known to be produced by common maize and maize-silage contaminants. A simple pH-buffered sample extraction was developed on the basis of a very fast and simple method for analysis of multiple pesticide residues in food known as QuEChERS. The buffering effectively ensured a stable pH in samples of both well-ensiled maize (pH < 4) and of hot spots with fungal infection (pH > 7). No further clean-up was performed before analysis using liquid chromatography-tandem mass spectrometry. The method was successfully validated for determination of eight analytes qualitatively and 19 quantitatively. Matrix-matched calibration standards were used giving recoveries ranging from 37% to 201% with the majority between 60% and 115%. Repeatability (5-27% RSD(r)) and intra-laboratory reproducibility (7-35% RSD(IR)) was determined. The limit of detection (LOD) for the quantitatively validated analytes ranged from 1 to 739 microg kg(-1). Validation results for citrinin, fumonisin B(1) and fumonisin B(2) were unsatisfying. The method was applied to 20 selected silage samples and alternariol monomethyl ether, andrastin A, alternariol, citreoisocoumarin, deoxynivalenol, enniatin B, fumigaclavine A, gliotoxin, marcfortine A and B, mycophenolic acid, nivalenol, roquefortine A and C and zearalenone were detected.

Development of an LC-MS/MS method for the determination of pesticides and patulin in apples.

A method for the simultaneous determination of 33 pesticides or degradation products together with patulin in apples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method involved homogenization of the apples, extraction with ammonium acetate-acetic acid solution in methanol-water by ultrasonication, filtration, and determination by LC-MS/MS. The repeatability and within-laboratory reproducibility for the three spiking levels 0.02, 0.04 and 0.2 mg kg(-1) were between 4% and 35%. In general, the repeatability and reproducibility were about 10-20%. The limits of quantification (LOQs) were between 0.01 and 0.14 mg kg(-1). The method was used on incurred samples from parts of the ISAFRUIT project financed by the European Commission under the 6th Framework Programme. Samples were analysed at four different stages: after harvest, after storage (controlled), after a water bath, and after 28 days at room temperature. Pesticide residues were found at all stages, but no significant differences in the concentration were seen between the stages analysed. The concentration decreased significantly only for tolylfluanid after storage at room temperature for 28 days when only 0-6% of the original amount of tolylfluanid remained in the apples. No patulin was found in the apples stored for 28 days at room temperature and no growth of Penicillium expansum was observed on these apples. However, when the apples were inoculated with a spore suspension of P. expansum, high concentrations of patulin were found.

Penicillium verrucosum occurrence and ochratoxin A contents in organically cultivated grain with special reference to ancient wheat types and drying practice.

This study addresses the relationship between the ochratoxigenic strains of Penicillium verrucosum and ochratoxin A (OTA) contents in organically cultivated grain. It included 37 combined, non-dried grain samples from farmers with no drying facilities as well as 19 non-dried and 22 dried samples from six farms with on-farm drying facilities (Case studies 1-6). The study focused on the ancient wheat type spelt but also included samples of wheat, rye, barley, oats, triticale, emmer, and einkorn. All 78 samples were analysed for moisture content (MC) and occurrence of P. verrucosum. The latter was assessed by plating non-disinfected kernels on DYSG agar and counting those contaminated by the fungus. Fifty-five samples were analysed for OTA. Most of the combine harvested samples (82%) were contaminated with P. verrucosum prior to drying. This was ascribed to difficult harvest conditions and many samples of spelt, which was significantly more contaminated by P. verrucosum than oats, wheat and barley. Though not statistically significant, the results also indicated that spelt was more contaminated than rye, which is usually regarded the most sensitive small grain cereal. No correlation was found between number of kernels contaminated by P. verrucosum and OTA content. Despite many non-dried samples being contaminated by P. verrucosum, only two exceeded the EU maximum limit for grain (5 ng OTA g(-1)), both being spring spelt with 18 and 92 ng g(-1), respectively. The problems were most likely correlated to a late harvest and high MC of the grain. The case studies showed exceedings of the maximum limit in a batch of dried oats and spring wheat, respectively, probably to be explained by insufficient drying of late harvested grain with high MC. Furthermore, our results clearly indicate that OTA is not produced in significant amounts in samples with MCs below 17%. All dried samples with MCs above 18% exceeded the 5 ng OTA g(-1) limit in grain. However, no correlation between MC and the amount of OTA produced was found.

Deoxynivalenol and other Fusarium toxins in wheat and rye flours on the Danish market.

Information on the contamination of Danish cereals and cereal products with Fusarium toxins is limited and the last survey is from 1984/1985. In the present study, the occurrence of deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and zearalenone (ZON) was investigated in flour of common wheat, durum wheat and rye. The samples were collected from 1998 to 2001 from both mills and the retail market in Denmark. A total of 190 flour samples were analysed for DON and NIV and about 60 samples for HT-2, T-2 toxin and ZON. DON was most frequently detected with an incidence rate of 78% over all samples for all years. The contamination level varied considerably from year to year, and for wheat and rye the highest incidence and DON concentrations were found in samples from the 1998 harvest. There were regular and heavy rainfalls in Denmark during the flowering period of the crops that year, and DON was found in all samples, with mean concentrations in wheat and rye flour of 191 microg kg(-1) (n=14) and 99 microg kg(-1) (n=16), respectively. Comparison of data from each harvest year showed higher contents of DON in samples of wheat (range 20-527 microg kg(-1)) than in rye (20-257 microg kg(-1)). Contents of NIV, HT-2 toxin and ZON in samples of wheat and rye were generally low, and even in positive samples the contents were close to the detection limit of the methods. The T-2 toxin was detected in only a few of the wheat samples and in low amounts. However, the toxin was found in about 50% of the rye samples collected during 1998-2000, with a mean content of 49 microg kg(-1) (n=25). Durum wheat flour showed the highest DON contamination level, and all samples (n=33) collected during 2000 and 2001 contained DON with means and medians above 1100 microg kg(-1). Over 70% of the samples contained more than 500 microg kg(-1) DON, and the highest observed concentration was 2591 microg kg(-1). The concentration of T-2 toxin in durum wheat flour was also high with five of the 10 analysed samples containing more than 100 g kg(-1).

Glycaemic index of parboiled rice depends on the severity of processing: study in type 2 diabetic subjects.

OBJECTIVE: To study the influence of parboiling and the severity of the process on glycaemic and insulinaemic responses to rice in type 2 diabetes. Moreover, to examine changes in starch structure related to parboiling, which may affect the metabolic responses and digestibility. DESIGN: Nine type 2 diabetic subjects ingested four test meals: white bread (WB) and three meals of cooked polished rice of the same variety being non-parboiled (NP), mildly traditionally parboiled (TP) and severely pressure parboiled (PP). The participants ingested the test meals (50 g available carbohydrates) on separate occasions after an overnight fast. SETTING: Outpatient clinic, Dept. Endocrinology and Metabolism, Aarhus University Hospital, Denmark. RESULTS: All three rice samples elicited lower postprandial plasma glucose response (NP: 335+/-43; TP: 274+/-53; PP: 231+/-37 mmol/1*180 min.; means+/-s.e.m.) than white bread (626+/-80; P<0.001), within rice samples PP tended to be lower than NP (P=0.07). The glycaemic indices were: NP: 55+/-5, TP: 46+/-8 and PP: 39+/-6, and lower for PP than NP (P<0.05). The insulin responses were similar for the three rice meals, which were all lower than that to white bread (P<0.001). Differential scanning calorimetry showed the presence of amylose-lipid complexes in all rice samples and of retrograded amylopectin in PP. Amylose retrogradation was not detected in any of the rice samples. CONCLUSIONS: All rice test meals were low-glycaemic in type 2 diabetic subjects. There was no effect of TP on glycaemic index, whereas PP reduced the glycaemic index by almost 30% compared to NP. SPONSORSHIP: The Royal Veterinary and Agricultural University, Aarhus University Hospital, Danish International Development Assistance (DANIDA), Ministry of Foreign Affairs and the 'Konsul Johannes Fogh-Nielsens og Fru Ella Fogh-Nielsens Legat' foundation.

Radioimmunoassay of endothelin in human plasma.

A specific and sensitive radioimmunoassay (RIA) for determination of endothelin-1 (ET-1) in human plasma has been developed. Antibodies were raised in rabbits using synthetic ET-1 conjugated to thyroglobulin as immunogen. The antibodies obtained were used at a final dilution of 1:300,000 yielding maximum binding of 61.7 +/- 3.0% (mean +/- 1 SD, n = 20) of 125I-ET-1. The ID50 (inhibitory dose 50%) was 4.5 +/- 0.6 fmol/100 microliters (mean +/- 1 SD, n = 20). The sensitivity of the RIA was 0.33 fmol/100 microliters standard solution. No cross reactivity was observed with endothelin-3, big-endothelin-1, atrial natriuretic factor, angiotensin I or angiotensin II. The cross-reactivity with endothelin-2 was 100%. Endothelin was extracted from acidified plasma with Sep-pak C18 cartridges and recovery of ET-1 added to normal plasma was 70.9 +/- 10.3% (mean +/- 1 SD, n = 12). The concentration of ET-1 in plasma from normal subjects was 1.5 +/- 0.4 pmol/l (mean +/- 1 SD, n = 11) ranging from 1.0 to 2.2 pmol/1. Extracts of normal human plasma subjected to high performance liquid chromatography on a reverse phase C18 column showed one peak of immunoreactivity co-eluting with the standard for ET-1. From these data it is concluded that the immunoreactive material measured in normal plasma with the present RIA is identical to ET-1.

Comparison of different standards used in radioimmunoassay for atrial natriuretic factor (ANF).

Six different standards for determination of atrial natriuretic factor (ANF) in human plasma samples have been compared using our radio-immunoassay for ANF: International standard 85/669, National Biological Standard Boards, UK; Bachem standard, Torrance, USA; Bachem standard, Bubendorf, Switzerland; Bissendorf standard, Wedemark, Germany; Peninsula standard, Belmont, USA; UCB-Bioproducts standard, Brussels, Belgium. Standard curves obtained with different preparations were in parallel but showed considerable quantitative differences. Standard curves referring to the Bissendorf standard and the International standard, respectively, were almost identical. The dose required for 50% of binding inhibition (ID50s) determined with the Peninsula, UCB and Swiss Bachem standards were higher and ID50 for the American Bachem standard was much lower than ID50 for the International standard. In consequence, estimates of the ANF content in human plasma samples with different standard preparations as the reference showed a considerable variability. With the international standard as the gold reference (plasma ANF concentration 100%) the apparent plasma ANF concentrations measured with the other reference preparations varied from 42% to 178%.

Solid-phase double-antibody radio-immunoassay for atrial natriuretic factor.

A solid-phase radio-immunoassay for the determination of atrial natriuretic factor (ANF) in human plasma is described. Iodination of alpha hANF was carried out with the iodogen method. Purification of radio-iodinated alpha hANF was performed by chromatography on disposable columns of DEAE-Sephadex A-25. Studies of immunoreactivity and the elution pattern on HPLC showed perfect stability of the labelled compounds. The tracer was usable for 28 weeks after preparation, and the batch-to-batch variation in the quality of the tracer was satisfactory. Immunoreactive ANF was extracted from human plasma with Sep-Pak C18 cartridges. Recovery of alpha hANF added to whole blood was 85 +/- 12% (mean +/- SD, n = 12). The sensitivity of the radio-immunoassay was 1.6 pg/tube, equivalent to 1 pg/ml plasma when assaying the extract from 4 ml plasma. Mean plasma ANF values in normal subjects in the supine position was 23 +/- 12 pg/ml (mean +/- SD, n = 21).

Direct and indirect blood pressure during exercise.

In 27 subjects, we compared rest and exercise blood pressure (BP) measurements determined directly by catheterization of the radial artery with simultaneous values obtained indirectly by auscultation of the brachial artery. As work increased, the systolic BP increased, whereas the diastolic BP did not change. Considering all comparisons, direct BP was greater than indirect BP by a mean of 29.0 mm Hg for systolic BP and 12.3 mm Hg for diastolic BP. As exercise level increased, the difference between direct and indirect systolic BP decreased whereas the difference between direct and indirect diastolic BP did not change. Both methods have advantages for assessment of BP response to exercise: normality of BP response is best assessed by auscultation, whereas beat-by-beat trends in BP are more accurately defined by the direct method.