The presence of ochratoxin A in cord serum and in human milk and its correspondence with maternal dietary habits.

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin present in food that can be found in human blood and milk. PURPOSE: The link between the nutritional habits of pregnant women both of Italian and foreign nationality resident in Italy and the presence of ochratoxin A in cord blood and in maternal milk was investigated. METHODS: The study involved 130 pregnant women. Food consumption during pregnancy was evaluated by means of the EPIC questionnaire; OTA content was determined in cord serum and maternal milk by HPLC. RESULTS: The mean daily dietary intake of OTA was 1.02 ± 1.20 and 0.87 ± 0.78 ng/kg of bodyweight for Italian and non-Italian women, respectively, but this difference was not statistically significant. The incidence of positive milk samples was 73.0 and 85.0% among the Italian and non-Italian mothers, respectively. Pork meat, soft drinks, sweets and red wine showed a significant relationship with OTA level in serum. As far as milk is concerned, a positive relationship resulted for pork meat, sweets, soft drinks and seed oils. A positive relationship between serum OTA level and the ratio serum/milk OTA was found. The intake of OTA had no effect on the cord blood creatinine level. CONCLUSIONS: This study confirms that OTA is widely present in human milk and therefore could pose a risk for the newborn.

Studies on Aspergillus section Flavi isolated from maize in northern Italy.

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.

Black aspergilli and ochratoxin A in grapes in Italy.

The objective of this research was to investigate the presence of black aspergilli in grapes grown in Italy and to study the effect of environmental and cultural factors able to influence fungal incidence and ochratoxin A (OTA) presence. In this 3-year study, black aspergilli were frequently associated with grape berries; they were present in bunches starting from setting, colonising most berries at early veraison. Aspergillus carbonarius was never dominant at the different growth stages, or in different geographic areas and years, but it was confirmed as the key fungus because of the high percentage of strong OTA producer isolates in the population. The number of OTA producer strains, isolated in each vineyard at the different growth stages, was generally very limited and they were never statistically correlated to OTA content in bunches. The effect of geographic area on fungal flora was confirmed by statistical analysis, even though a major role was played by meteorological conditions, both on fungal colonisation and OTA content in bunches. Discriminant analysis gave promising perspectives for predicting OTA presence in vineyards in the future, based on summation of degree-day and rain in the period between 21st of August and 10th of September.

Changes in glucagon levels after four to five weeks of glucoregulation by portable insulin infusion pumps.

Near-normal glucoregulation was maintained in five patients with juvenile-onset diabetes mellitus for 4--5 wk with a preprogrammed, continuous, subcutaneous insulin infusion using a portable battery-powered infusion pump. This form of therapy significantly lowered immunoreactive glucagon (IRG) levels below those observed while on conventional insulin treatment at several times during the 24-h profile. The maximum IRG levels were also reduced in all five subjects. Thus, a flexible system of insulin delivery, as is provided by certain open-loop pump systems, can overcome inappropriate glucagon secretion that occurs with conventional insulin therapy.

The effect of improved diabetic control on plasma lipid and lipoprotein levels: a comparison of conventional therapy and continuous subcutaneous insulin infusion.

We studied short-term changes in plasma lipid levels in type I diabetics treated with either a conventional insulin regimen or continuous subcutaneous insulin infusion. Mean plasma glucose dropped from 260 +/- 18 to 134 +/- 8 mg/dl when conventional treatment was used and from 194 +/- 18 to 108 +/- 8 mg/dl with CSII. Both forms of therapy were associated with a significant fall in plasma triglyceride levels. However, only CSII treatment produced significant changes in total plasma cholesterol and LDL cholesterol levels. Total cholesterol fell from 195 +/- 17 mg/dl to 161 +/- 11 mg/dl and LDL cholesterol fell from 129 +/- 13 mg/dl to 102 +/- 9 mg/dl. We conclude that improved diabetic control by any method is effective in lowering plasma triglyceride levels, but it requires almost perfect metabolic control to affect plasma cholesterol and LDL cholesterol levels. The changes in plasma lipid and lipoprotein achieved with CSII may favorably alter the prediction for the development of premature atherosclerosis in our patients.

Changes in nerve conduction velocity after six weeks of glucoregulation with portable insulin infusion pumps.

Near normal glucoregulation was maintained in 10 patients with insulin-dependent (type I) diabetes mellitus for 6 wk with preprogrammed continuous subcutaneous insulin infusion using a portable battery-powered infusion pump (CSII). This form of therapy resulted in a statistically significant increase in motor nerve conduction velocity in the median and peroneal nerves compared with baseline values. There was no significant change in the motor nerve conduction velocity in the ulnar nerve or in the sensory nerve conduction studies. No changes occurred in five additional patients studied in similar fashion while on a conventional insulin regimen. These results suggest that the prevention of sustained hyperglycemia with CSII could theoretically result in the prevention of diabetic neuropathy. However, only long-term studies of CSII will provide the information necessary to determine the clinical relevance of the findings.

Evidence for the hormonal status of somatostatin in man.

To determine the hormonal status of somatostatin in man, plasma levels of approximately 1600-dalton somatostatin-like immunoreactivity (SLI) were measured before and after a mixed meal. Plasma was subjected to gel filtration and the fractions coeluting with somatostatin were concentrated ninefold before radioimmunoassay. In this system the recovery of synthetic somatostatin added to plasma averaged 71 +/- 4.6% (mean +/- SE, N = 5). Synthetic somatostatin infused into normal volunteers gave a dose-related increase in the measured SLI value. Fasting SLI in 13 normal volunteers was 8.0 +/- 0.7 pg/ml (mean +/- SE) and rose to 18.6 +/- 1.5 pg/ml and 20.3 +/- 2.0 pg/ml at 60 and 120 min, respectively, after the meal (P less than 0.001). In seven type I diabetics, the fasting level was 11.5 +/- 1.6 pg/ml and the 60- and 120-min postprandial levels were 21.4 +/- 2.5 and 22.3 +/- 2.9 pg/ml, respectively. The meal-induced rise in approximately 1600 M.W. SLI approximated that produced by infusing somatostatin at 2 microgram/h, a rate that significantly suppressed glucagon levels. These results are therefore consistent with a hormonal role for somatostatin in man.

Effect of Bt corn on broiler growth performance and fate of feed-derived DNA in the digestive tract.

The aim of the study was to evaluate the effect on broiler performance of transgenic Bacillus thuringiensis (Bt) corn containing the Cry1A(b) protein compared with the corresponding near isogenic corn and to analyze the degradation of the Cry1A(b) gene in the digestive tract. Ross male broilers (432) were fed for 42 consecutive days with diets containing Bt or isogenic corn. Diet, Bt corn, and the isogenic form of the Bt corn were analyzed for composition and aflatoxin B1, fumonisin B1, and deoxynivalenol contents. Broiler body weight and feed intake were recorded at regular intervals (d 0, 21, and 42). The presence of the Cry1A(b) gene and plant-specific genes Zein and Sh-2 in gut contents of crop, gizzard, jejunum, cecum, and samples of blood was determined in 10 animals per treatment at the end of the trial using a PCR technique. Chemical composition was not different between Bt and its isogenic form, whereas the fumonisin B1 content for Bt was lower than for isogenic corn (2,039 vs. 1,1034 ppb; P < 0.05). The results of the growth study showed no difference for average daily weight gain (129.4 vs. 126.0 g/d), feed intake (63.4 vs. 61.8 g/d), and feed conversion ratio (1.95 vs. 2.02) among the groups. No significant relationship was observed between mycotoxins content and growth performances. Feed-derived DNA is progressively degraded along the digestive tract. Detection frequency of short fragments of maize-specific high copy number Zein gene was high but significantly decreased in distal sectors. An 1,800-bp fragment of the Cry1A(b) gene, corresponding to the minimal functional unit, was detected only in crop and gizzard of birds fed Bt corn. Sh-2 showed the same detection frequency of Cry1A(b) and was also found in birds fed isogenic corn. Blood samples were positive with low frequency only for the Zein gene fragment. No significant difference in DNA detection was observed between birds fed Bt and isogenic corn, indicating that DNA derived from transgenic feed undergoes the same fate as isogenic feed.

Cutaneous complications of chronic continuous subcutaneous insulin infusion therapy.

The cutaneous complications that can develop with continuous subcutaneous insulin infusion constitutes a significant concern when using this innovative and highly effective form of treatment for type I diabetes. These complications are twofold, the first being infection with abscess formation at the infusion needle site. Proper hygiene and frequent changing of the infusion site are helpful and effective measures. The second complication is the development of local allergic skin reactions, again at the infusion needle site, even with highly purified pork insulin. These may subside after several weeks on their own but switching insulin preparations also may be helpful.

The effect of continuous subcutaneous insulin infusion on endogenous insulin secretion in type I diabetes.

In an attempt to elucidate possible mechanisms for the success of continuous subcutaneous insulin infusion (CSII), we evaluated the C-peptide response to a standard breakfast in seven type I diabetic patients while they were on conventional insulin treatment and again after 4 wk of near-normal glycemia achieved with CSII. While on conventional therapy their 24-h mean blood glucose level was 211 +/- 12 mg/dl and their glycosylated hemoglobin level was 10.6 +/- 0.6%. After 4 wk of CSII their 24-h mean blood glucose level fell to 95 +/- 7 mg/dl and their glycosylated hemoglobin level fell to 6.5 +/- 0.4%. Plasma C-peptide levels were undetectable in all seven patients both while on conventional therapy and after 4 wk of CSII. We conclude that the success of CSII is related to an improved method of insulin delivery and not to either the selection of type I diabetic patients who have some residual insulin secretory capacity or to some change in endogenous insulin secretion produced by the treatment itself.

Improved control of mealtime glucose excursions with coadministration of nateglinide and metformin.

OBJECTIVE: Nateglinide, a new short-acting D-phenylalanine derivative for treating type 2 diabetes, reduces mealtime blood glucose excursions by physiologic regulation of insulin secretion. This study evaluated the pharmacokinetic and pharmacodynamic interactions of nateglinide and metformin in subjects with type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 12 type 2 diabetic subjects with the following baseline characteristics were enrolled: age, 56 +/- 13 years; BMI, 28.7 +/- 4.5 kg/m2; HbA1c, 8.4 +/- 1.3%; and fasting plasma glucose 13 +/- 2.8 mmol/l. All subjects had been previously treated with glyburide and were switched to metformin monotherapy for 3 weeks before study start. Subjects then randomly received, in combination with 500 mg metformin, either 120 mg nateglinide or placebo before meals for 1 day, followed by the alternate treatment 7 days later. After 1 week of washout from both drugs, subjects received 1 day of open-label nateglinide treatment. Plasma concentrations of glucose, insulin, nateglinide, and metformin were assessed frequently during inpatient periods. RESULTS: Postmeal plasma glucose levels were significantly lower in subjects treated with nateglinide plus metformin than in those treated with either drug alone (P < 0.001), especially after lunch and dinner. Coadministration of nateglinide and metformin did not affect the pharmacokinetics of either drug. All treatments were safe and well tolerated. CONCLUSIONS: Combination therapy with nateglinide and metformin was more effective than either treatment alone and did not result in any pharmacokinetic interactions. Coadministration of nateglinide and metformin appears to be an excellent option for treating patients with type 2 diabetes not controlled with monotherapy.

Ineffectiveness of activated carbon in reducing the alteration of sphingolipid metabolism in rats exposed to fumonisin-contaminated diets.

Sphinganine/sphingosine (SA/SO) ratio, a biomarker of fumonisin exposure, has been measured in urine, kidney and liver of male Wistar rats exposed to fumonisin-contaminated diet with and without the addition of activated carbon (AC). The latter was previously shown to adsorb fumonisin B(1) in vitro. Rats were fed either control diet or fumonisin-contaminated diet (4 microg FB(1+)FB(2)/g) or fumonisin-contaminated diet mixed with 20 mg AC/g diet for 1 week. In rats fed fumonisin-contaminated diet, the SA concentration and SA/SO ratio increased significantly and reversibly in kidney, while urine and liver did not show a significant increase of SA/SO ratio. The addition of AC to the fumonisin-contaminated diet did not alter the change of SA/SO biomarker for fumonisin exposure. This provides indications that the use of AC to reduce the toxicity of fumonisins is unlikely to be effective in vivo.

Activated carbons: in vitro affinity for ochratoxin A and deoxynivalenol and relation of adsorption ability to physicochemical parameters.

In vitro affinity tests were conducted to test the effectiveness of 19 activated carbons (ACs), hydrates sodium calcium aluminosilicate (HSCAS) and sepiolite (S) in binding ochratoxin A (OA) and deoxynivalenol (DON) from solution. Relationships between adsorption ability and physicochemical parameters of ACs (surface area, iodine number, methylene blue index) were tested. When 5 ml of a 4-micrograms/ml aqueous solution of OA was treated with 2 mg of AC, the ACs adsorbed 0.80 to 99.86% of the OA. HSCAS and S were not effective in binding OA. In two saturation tests carried out with increased amounts of OA (5 ml of 10-and 50-micrograms/ml aqueous solutions of OA, respectively) three ACs also showed high adsorption ability (adsorbing 92.23 to 96.57% of the OA). When 5 ml of a 4-micrograms/ml aqueous solution of DON was treated with 10 mg of AC, ACs adsored 1.83 to 98.93% of the DON. HSCAS and S were not effective in binding DON. An overall relation of adsorption ability to the physicochemical parameters of ACs was observed. The methylene blue index was more reliable than iodine number and surface area in predicting ability of ACs to adsorb OA and DON. Based on the data observed on the xxxxx eh present study as well as on aflatoxin B1 and fumonisin B1 from previous studies, it is concluded that ACs have high in vitro affinity for chemically different mycotoxins, and can be considered as potential multi-mycotoxin-sequestering agents. However, the ability to bind the main mycotoxins singly or in combination should be confirmed by in vivo investigations. Moreover, information on the amounts of AC to be added to feeds, and on the possible long-term effect on absorption of essential nutrients are needed.

Evaluation of a fluorodensitometric method for analysis of ergosterol as a fungal marker in compound feeds.

Ergosterol is the principal sterol of fungi and plays an essential role as a component of the cell membrane and other cell constituents. This molecule is considered a good marker of fungal contamination in foods and feeds. This paper reports a rapid and sensitive method to test ergosterol content in compound feeds based on fluorodensitometry after thin-layer chromatography (TLC) separation. This method involves a thermal treatment of TLC plates that leads to the formation of a highly fluorescent ergosterol derivative. Such a dosage allows ergosterol testing in any naturally contaminated samples (limit of detection: 1 ppm of ergosterol) and gives results in close agreement with high-pressure liquid chromatography determination. Moreover, values obtained on mixed feeds for animals at different steps of fungal contamination are linked to quantitative development of storage fungi, evaluated by mycological technique, reinforcing the interest of a rapid method for measuring this fungal marker.

Enzyme-assisted extraction for the HPLC determination of ochratoxin A in pork and dry-cured ham.

The extraction of ochratoxin A from meat products is generally carried out using chlorinated organic solvents, such as chloroform or methyl chloride, acidified with hydrochloric or o-phosphoric acid. In this study, an innovative method was developed to extract ochratoxin A from pork and dry-cured ham samples. The method was based on an enzyme-assisted extraction with pancreatin in phosphate buffer pH 7.5. Pancreatin hydrolyses the proteins, so that ochratoxin A, kept in the ionised form, is easily extracted by the aqueous solution. After purification through an immunoaffinity column, ochratoxin A is determined by HPLC with fluorescence detection. The average recovery values were higher than 90.0% and the relative standard deviations were below 5.5%. The limits of detection and of quantification were 0.06 and 0.12 µg kg(-1), respectively. A comparison between the new enzyme-assisted extraction and an established chloroform method was carried out on six naturally contaminated samples of pork and on 40 samples of dry-cured ham. Significantly higher (p<0.001) values of ochratoxin A were obtained on dry-cured ham samples by the enzyme-assisted method.

Transfer of aflatoxin B1 and fumonisin B1 from naturally contaminated raw materials to beer during an industrial brewing process.

The aim of this research was to determine the fate of aflatoxins (AFs) and fumonisins (FBs) naturally occurring in raw materials (maize grit and malted barley) during four industrial brewing processes. The aflatoxin B(1) (AFB(1)) level in raw materials varied from 0.31 to 14.85 microg kg(-1), while the fumonisin B(1) (FB(1)) level (only in maize grit) varied from 1146 to 3194 microg kg(-1). The concentration in finished beer ranged from 0.0015 to 0.022 microg l(-1) for AFB(1) and from 37 to 89 microg l(-1) for FB(1); the other aflatoxins and fumonisin B(2) were not found in beer samples. The average percentage of toxins recovered in finished beer, referring to the amounts contained in raw materials, were 1.5% +/- 0.8% for AFB(1) and 50.7% +/- 4.7% for FB(1). These results were mainly due to the different solubility of the two mycotoxins during the mashing process. If raw materials comply with the limits fixed by European Commission Regulations, the contribution of a moderate daily consumption of beer to AFB(1) and FB(1) intake does not contribute significantly to the exposure of the consumer.

Effect of the inclusion of adsorbents on aflatoxin B1 quantification in animal feedstuffs.

The extraction efficiency of aflatoxin B1 (AFB1) in cattle feed containing nine adsorbents (ADSs) was investigated using two organic/aqueous solvents composed of methanol/water (80/20 v/v; MeOH) and acetone/water (85/15 v/v; AC). Samples were obtained including a highly AFB1-contaminated (HC) and a low-level AFB(1)-contaminated (LC) feedstuff (15.33 and 7.57 microg kg(-1), respectively), nine ADSs (four clay minerals; one yeast cell wall-based product; one activated carbon and three commercial ADS products) at two different levels of inclusion (10 and 20 g kg(-1)). After solvent extraction and immunoaffinity column clean-up, all samples were analysed for AFB1 by high-performance liquid chromatography (HPLC) with fluorescence detection. For each contamination level (HC and LC), the data obtained were analysed using a factorial arrangement in a completely randomized design. Means were compared with the correspondent controls using the Dunnett's test. No statistical difference was found in AFB1 levels of feedstuffs not containing ADSs when extracted with AC or MeOH, even if numerically higher values were obtained with AC. A dose-dependent effect (p < 0.01) of ADSs inclusion was observed on AFB1 recoveries that were lower when the higher ADS level (20 g kg(-1)) was included in the HC and LC feedstuffs. Higher AFB(1) recoveries were obtained using AC compared with MeOH, both in HC (75.0% versus 12.0%, respectively) and in LC (84.0% versus 22.8%, respectively) ADSs containing feedstuffs. However, when the activated carbon and the sodium bentonite were included in feeds, lower AFB1 concentrations with respect to control values (p < 0.001 and <0.05, respectively) were obtained also using AC. The data obtained in this study indicate that routine use of the MeOH solvent for AFB1 analysis of unknown feedstuffs, can produce misleading results if they contain an ADS.