Aflatoxin and ochratoxin A content of spices in Hungary.

In October and November 2004, 91 spice samples (70 ground red pepper, six black pepper, five white pepper, five spice mix and five chilli samples), the majority of which originated from commercial outlets, were analysed for aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) content by high-performance liquid chromatography (HPLC) after immunoaffinity column clean-up. Eighteen of the 70 ground red pepper samples contained AFB1, seven of them in a concentration exceeding the 'maximum level' of 5 microg kg(-1) (range 6.1-15.7 microg kg(-1)). Of the other spices assayed, the AFB1 contamination of one chilli sample exceeded 5 microg kg(-1) (8.1 microg kg(-1)). Thirty-two of the 70 ground red pepper samples contained OTA, eight of them in a concentration exceeding the 10 microg kg(-1) 'maximum level' (range 10.6-66.2 microg kg(-1)). One chilli sample was contaminated with OTA at 2.1 microg kg(-1). The AFB1 and OTA contamination of ground red pepper exceeding the 'maximum level' (5 and 10 microg kg(-1), respectively) was obviously the consequence of mixing imported ground red pepper batches heavily contaminated with AFB1 and OTA with red pepper produced in Hungary. This case calls attention to the importance of consistently screening imported batches of ground red pepper for aflatoxin and ochratoxin A content and strictly prohibiting the use of batches containing mycotoxin concentrations exceeding the maximum permitted level.

Ochratoxin a contamination of cereal grains and coffee in Hungary in the year 2001.

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin, a secondary metabolite produced by mould fungi belonging to several Aspergillus and Penicillium species. It is formed during the storage of cereal grains and other plant-derived products. OTA ingested by humans and animals with the food or feed may exert deleterious effects on health. The purpose of this study was to investigate the ochratoxin contamination of the most important potential sources of OTA. The OTA content of cereal samples for human consumption (36 baking wheat, 16 wheat flour and 6 maize coarse meal samples) and feed grain samples (30 feeding wheat, 32 feeding maize and 20 feeding barley samples) collected in the mid-phase or at the end of the storage period and of 50 commercial coffee samples was determined. The analyses were performed by immunoaffinity column--high-performance liquid chromatography (IAC-HPLC). The limit of detection of the method was 0.1 ng/g. Of the wheat samples intended for human consumption, 8.3% contained OTA at 0.29 ng/g on the average (OTA ranges: 0.12-0.5 ng/g; Table 2). The OTA contamination of wheat flour and maize meal samples for human consumption was similar to that of the baking wheat samples. OTA contamination was found in 26.7% of the feeding wheat, 15.6% of the feeding maize and 35% of the feeding barley samples. The average values and the ranges of OTA levels found in the above samples were 12.2 and 0.3-62.8 ng/g, 4.9 and 1.9-8.3 ng/g, and 72 and 0.14-212 ng/g, respectively (Table 3). Sixty-six percent of the coffee samples were contaminated with OA (average level: 0.57 ng/g, ranges: 0.17-1.3 ng/g; Table 4). OTA contamination of baking wheat samples was found to be relatively low, presumably as a result of the favourable weather at harvest and the optimal storage conditions. Calculations made on the basis of the obtained results show that the daily OTA intake of an adult human from edible cereals is only 6.7 ng, while the amount taken up with coffee is 4.1 ng daily. The high prevalence and high levels of OTA contamination in feed grains can be explained by the unfavourable storage conditions, and this finding suggests that OA-related health problems may arise in animals, and that foods of animal origin may be contaminated with this mycotoxin.

Determination of zearalenone content in cereals and feedstuffs by immunoaffinity column coupled with liquid chromatography.

The zearalenone content of maize, wheat, barley, swine feed, and poultry feed samples was determined by immunoaffinity column cleanup followed by liquid chromatography (IAC-LC). Samples were extracted in methanol-water (8 + 2, v/v) solution. The filtered extract was diluted with distilled water and applied to immunoaffinity columns. Zearalenone was eluted with methanol, dried by evaporation, and dissolved in acetonitrile-water (3 + 7, v/v). Zearalenone was separated by isocratic elution of acetonitrile-water (50 + 50, v/v) on reversed-phase C18 column. The quantitative analysis was performed by fluorescence detector and confirmation was based on the UV spectrum obtained by a diode array detector. The mean recovery rate of zearalenone was 82-97% (RSD, 1.4-4.1%) on the original (single-use) immunoaffinity columns. The limit of detection of zearalenone by fluorescence was 10 ng/g at a signal-to-noise ratio of 10:1 and 30 ng/g by spectral confirmation in UV. A good correlation was found (R2 = 0.89) between the results obtained by IAC-LC and by the official AOAC-LC method. The specificity of the method was increased by using fluorescence detection in parallel with UV detection. This method was applicable to the determination of zearalenone content in cereals and other kinds of feedstuffs. Reusability of immunoaffinity columns was examined by washing with water after sample elution and allowing columns to stand for 24 h at room temperature. The zearalenone recovery rate of the regenerated columns varied between 79 and 95% (RSD, 3.2-6.3%). Columns can be regenerated at least 3 times without altering their performance and without affecting the results of repeated determinations.

Novel AIRE mutations and P450 cytochrome autoantibodies in Central and Eastern European patients with APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare recessive disorder that results in several autoimmune diseases due to the mutations in the AIRE (autoimmune regulator) gene. APECED patients develop several autoimmune endocrine disorders and are characterized by the high titer autoantibodies to organ-specific antigens such as the steroidogenic P450 cytochromes. So far, 38 mutations have been identified in the AIRE gene. We report here the genetic and autoantibody analysis of 27 APECED patients of Eastern and Central European origins and one Egyptian patient. From 54 analyzed APECED chromosomes, eight mutations were detected, four of which (T16M, W78R, IVS1_IVS4, 30-53dup23bp) are novel. The most prevalent reason for APECED in these populations was the occurrence of R257X (36 chromosomes) that has been described earlier as a common and recurrent mutation in several other populations. The analysis of humoral immunity to steroidogenic P450 cytochromes by the immunoblotting of E. coli expressed antigens in the 18 APECED patients showed that 67%, 44%, and 61% of the Eastern and Central European APECED patients had autoantibodies to P450c17, P450c21, and P450scc, respectively.

Natural deoxynivalenol (DON) contamination of wheat samples grown in 1998 as determined by high-performance liquid chromatography.

A high-performance liquid chromatography--diode array detection (HPLC-DAD) method was developed for determining the deoxynivalenol (DON) content of wheat and other cereals. The samples were extracted with a mixture of acetonitrile and water (84 + 16). Part of the extract was evaporated and purified on Florisil and activated charcoal columns. HPLC separation was performed on a C18 column, using acetonitrile-water (8 + 92) as eluent. Diode array detection (DAD) was performed at 218 and 236 nm, by determination of the UV spectrum. Quantitative analysis was carried out by the external standard method, using the UV spectrum obtained by DAD for confirmation. The recovery rate of DON was 75 +/- 3.1% and the detection limit was 0.05 mg/kg DON. Using this method, the DON content of 99 feeding wheat samples grown in the northeastern part of Hungary in 1998 was determined. Eighty-eight percent of the samples originating from three counties contained 0.94 mg/kg DON on the average. The highest individual value was 4.3 mg/kg. DON contamination of wheat was of higher prevalence (100%) and severity (0.27-4.3 mg/kg) in the southeastern county of Békés than in Szabolcs county located in the northeastern part of Hungary (ratio of positive samples: 82%; DON concentration: 0.05-1.3 mg/kg). The higher than usual DON contamination of feeding wheat can be explained by the rainy summer weather. DON contamination of feeding wheat poses a major risk to the production and animal health status of pig herds.

A high frequency of Y chromosome deletions in males with nonidiopathic infertility.

Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, and AZFc) that are recurrently deleted in infertile males. In a blind study we screened 131 infertile males (46 idiopathic and 85 nonidiopathic) for Y chromosome microdeletions. Nineteen percent of idiopathic males, with an apparently normal 46,XY chromosome complement had microdeletions of either the AZFa, AZFb, or AZFc region. There was no strict correlation between the extent or location of the deletion and the phenotype. The AZFb deletions did not include the active RBM gene. Significantly, a high frequency of microdeletions (7%) was found in patients with known causes of infertility and a 46,XY chromosome complement. These included deletions of the AZFb and AZFc regions, with no significant difference in the location or extent of the deletion compared with the former group. It is recommended that all males with reduced or absence sperm counts seeking assisted reproductive technologies be screened for deletions of the Y chromosome.

The same mutation affecting the splicing of WT1 gene is present on Frasier syndrome patients with or without Wilms' tumor.

Denys-Drash and Frasier syndromes are rare human disorders that associate nephropathy with gonadal and genital abnormalities. In DDS there is a predisposition to Wilms' tumor. Heterozygous point mutations in the Wilms' tumor, type1 gene (WT1), particularly those altering the zinc finger (ZF) encoding exons, have been reported in most DDS patients, while mutations in intron 9 of the same gene cause FS. This paper describes two cases of DDS, one FS and one patient with Wilm's tumor and intersex genitalia, in which mutations were searched by sequencing the exons 8 and 9 of WT1 gene. Patient 1 carried a missense point mutation in exon 8 (ZF2), converting a CGA-Arg codon to a TGA-stop codon. Patient 2 presented a single nucleotide deletion within exon 9 (ZF3) introducing a premature chain termination at codon 398. Patients 3 and 4 had a C-->T transition at position +4 of the second alternative splice donor site of exon 9 (this mutation was detected in peripheral blood and in tumor derived DNA of patient 3). However, patient 3 had previously developed a Wilms' tumor. This is the first case of Wilms' tumor development in a phenotypically and genetically confirmed case of FS.

Reusability of immunoaffinity columns for determination of fumonisins in maize.

Eighteen maize samples were assayed for fumonisin B1 (FB1) and B2 content by immunoaffinity column coupled with high performance liquid chromatography (HPLC). The FumoniTest columns were used once for the isolation of fumonisins (single-use column method). In the second part of the assay the columns were regenerated. After elution with methanol, PBS solution was left on the column for one day at room temperature to regenerate the columns (regenerated column method). The efficiency of columns regenerated twice was tested by determining FB, recovery and the reproducibility of the determinations. The recovery rate of FB1 proved to be 82% by the single-use column method (RSD: 5.7%) and 82.6% (RSD: 5.6 %) by the regenerated column method; 500-8,000 ng FB1 loaded onto the columns did not affect column performances. Nearly identical values were obtained when the FB1 content of fumonisin-containing maize samples was determined by both methods. The results indicate that the FumoniTest columns can be regenerated by the method applied at least twice without decrease in column performance. The fumonisin affinity, capacity and specificity of the regenerated columns were not changed. Thus, columns regenerated in this way can be used for determining the fumonisin content of maize samples at least three times.

Hypertelorism and hypospadias associated with a de novo apparently balanced translocation between 8q22.3-23 and 20p13.

A de novo apparently balanced translocation involving chromosomes 8 and 20 was found in a 14-year-old boy with minor anomalies, mild skeletal abnormalities and ambiguous external genitalia including perineoscrotal hypospadias, rudimentary fused labioscrotal folds, bilateral cryptorchidism, and small penis. The karyotype was 46,XY, t(8;20)(q22.3-23;p13). No signs of other conditions known to be associated with structural anomalies of either chromosome 8 or 20 were present and incomplete masculinisation of the external genitalia appears to be the main component of the phenotype. Clinical and biological studies showed apparently normal testicular function in utero and after birth. Examinations excluded 5 alpha-reductase deficiency or a block in any enzymatic steps of testosterone, glucocorticoid and mineralocorticoid biosynthesis. Coding sequences of the sex-determining gene (SRY) and androgen receptor gene (AR) were found to be identical to those of a normal male excluding their role in the cause of the present condition. Since several other reports describe the association of hypospadias and hypertelorism with deletions or translocations involving 8q, we suggest that a locus necessary for male sex differentiation is located at distal 8q.

[46,XX karyotype males, based on a specific case]. / A 46,XX karyotypusú férfiakról egy eset kapcsán.

Individuals with 46,XX karyotype and testicular tissue are known as XX males. They either have male phenotype or sexual ambiguity. SRY (testis determining factor) is present in 80% of the reported cases. In our patient the presence of SRY was verified by polymerase chain reaction, explaining the sex reversal.

Testicular development in an SRY-negative 46,XX individual harboring a distal Xp deletion.

A case of a true hermaphrodite presenting with a karyotype of 46,X,del(X)(p21.1-->pter) is described. The testis-determining gene, SRY, was not detected in DNA prepared from either peripheral blood lymphocytes or from a gonad biopsy. The patient also presented with a series of discrete somatic abnormalities, including abnormal skin and retinal pigmentation, and mental retardation. The extent of the Xp deletion was mapped by Southern blotting. X chromosome replication studies of lymphoblast cells prepared from the patient indicated that the deleted X chromosome was inactivated in all cells examined. It is suggested that the phenotype of the patient is caused by the unmasking of a recessive allele(s) on the grossly intact X chromosome. The relationship between the Xp deletion, the intersex phenotype, and the possible role of an Xp locus involved in human sex determination is discussed.

[Analysis of the relationship between the effect of growth hormone therapy and the width of the radial metaphysis]. / A növekedési hormon kezelés hatása és a radiális metaphysis szélessége közötti összefüggés elemzése.

A radio-dense metaphyseal band (MB) of different width is present at the distal end of the radius in growing children. Authors studied 30 prepubertal children before and after one and two years of treatment by recombinant human growth hormone (rhGH). They examined the relationship between the MB, bone age (BA), height velocity (HV) and insulin like growth factor-I (IGF-I). However all the four parameters showed significant change in the study period, correlation was found only after the second year of treatment between the change MB and IGF-I levels. Authors suggest that the regulation of bone growth in these children can be compared only after two years to the normally growing children.

[Analysis of human growth hormone-binding protein in plasma by high pressure liquid chromatography]. / A humán növekedési hormon kötöfehérjék vizsgálata plazmában magas nyomású folyadékkromatográfiával.

A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (GH-BPs) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microliters) was incubated with 125I-human (h) GH (200,000 cpm) at 4 degrees C for 20 hours. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.

[Determination of growth hormone binding protein in a normal population and in subjects with short stature due to growth hormone resistance]. / A növekedési hormon kötöfehérje meghatározása egészséges populációban és növekedési hormon rezisztenciával járó, alacsonynövés állapotaiban.

With the procedure which uses gel-filtration and high pressure liquid chromatography (HPLC) the authors measured the GH-binding proteins in 96 healthy subjects aged 6 months to 40 years. In the blood 45% of the GH is bound to the BP. The diagnosis was confirmed by our method in 13 patients with Laron type dwarfism and in four patients the GH receptor defect had been proved. The parents, the brothers and sisters showed significantly lower GH-BP II (principal GH-BP) levels. Possible regulation of GH-binding proteins in human plasma was examined. Eight children with isolated GH deficiency had a very low level of plasma GH binding activity (mean +/- SEM) (10.2 +/- 1.1% of radioactivity). Under GH treatment the hormone binding to high affinity BP (GH-BP II) increased in every patient to reach the mean value of 18.5 +/- 1.4%. This increase was related to a higher binding capacity without any significant change in the binding affinity. In nine boys with pubertal delay, the GH specific binding to peak II-BP (GH-BP II) was found to be normal (30.6 +/- 3.7%); it decreased significantly following testosterone treatment. In four boys with precocious puberty, the specific GH binding to peak II BP was low (16.6 +/- 1.1%). It increased significantly to 21.6 +/- 1.1% of radioactivity after treatment with LH-RH analogue. GH and testosterone have an opposite role in the regulation of the high affinity GH-BP.

Plasma growth hormone-binding activity is low in uraemic children.

Plasma growth hormone-binding protein (GH-BP) activity was evaluated in two groups of prepubertal children with chronic renal failure (CRF) who had been treated with recombinant human GH (rhGH). Group 1 consisted of eight children (mean chronological age 10.8 years) with advanced renal failure; group 2 consisted of nine children (mean chronological age 6 years) presenting with end-stage renal disease, who were on dialysis. Before treatment the specific binding of (125I)hGH to high-affinity GH-BP was low in the two groups (group 1, 17.3 +/- 1.6% of radioactivity; group 2, 14.2 +/- 1.4%) compared with the mean value obtained in normal prepubertal children (24.8 +/- 1.7%). No significant changes in GH-BP activity were found during the 1st year of GH therapy, although growth velocity and plasma levels of insulin-like growth factor-I increased significantly in both groups. The low GH-binding activity found in children with CRF supports the state of GH resistance. The reason for the absence of a GH-BP response to GH therapy has to be clarified.

Human plasma growth hormone (GH)-binding proteins are regulated by GH and testosterone.

Possible regulation of GH-binding proteins (GH-BPs) in human plasma was examined. Eight children with isolated GH deficiency had a very low level of plasma GH-binding activity (10.2 +/- 1.1% of radioactivity). Under GH treatment the hormone binding to the high affinity BP (peak II-BP) increased in every patient to reach the mean value of 18.5 +/- 1.4%. In one patient, Scatchard plot analysis indicated that this increase was related to a higher binding capacity without any significant change in the binding affinity. A positive correlation existed between the GH-binding activity and insulin-like growth factor-I plasma levels. In nine boys with pubertal delay, the GH-specific binding to peak II-BP was normal (30.6 +/- 3.7% of radioactivity); it decreased significantly after testosterone treatment. In four boys with precocious puberty, the specific GH binding to peak II-BP was low (16.6 +/- 1.1%). It increased significantly to 21.6 +/- 1.1% of radioactivity after treatment with a LHRH analog. A negative correlation existed between plasma testosterone levels and GH binding to peak II-BP in boys presenting pubertal delay or precocious puberty. The high affinity GH-BP is regulated, and among the factors that play a role in this regulation, GH and testosterone have opposite effects.

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028.

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.

Regulation of the growth hormone binding proteins in human plasma.

The plasma high affinity growth hormone binding protein corresponds to the extracellular binding domain of the liver membrane receptor. A distinct higher molecular weight protein, which binds GH with low affinity and high capacity, is present in the human plasma. In man, the biosynthesis and the exact functions of the GH binding proteins have to be clarified. The GH binding proteins are differently regulated; they are under a multihormonal control. The high affinity binding protein is low in neonates, increases after the first year of life, and reaches its highest value in young adult, without sex differences. GH is able to induce the high affinity binding protein, whereas sex steroids can decrease it. States of GH resistance, such as chronic renal failure, and certain idiopathic short statures, are associated with low levels of GH binding protein; in Laron-type dwarfism a defect of the GH receptor gene probably results in the absence of plasma GH binding activity. Recent findings on the regulation of the GH binding protein in man support a parallel regulation of liver membrane GH receptors and plasma binding protein.

Evaluation of the growth hormone-binding proteins in human plasma using high pressure liquid chromatography gel filtration.

A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (BP) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microL) was incubated with [125I]human (h) GH (200,000 cpm) at 4 C for 20 h. The main GH-BP (peak II) was well separated from free [125I]hGH (peak III) and from a higher mol wt complex (peak I), which was minor. In our control plasma, the specific binding of [125I]hGH to peak II BP (II-BP) was 32.2 +/- 0.6% of the radioactivity. Scatchard analyses indicate an association constant of 3.6-7.4 X 10(8) M-1 and a binding capacity ranging from 24-86 ng/mL for peak II-BP in five normal adult plasma samples. Peak I material, separated from plasma of boys with pubertal delay, bound hGH with a low affinity (3 x 10(6) M-1) and a very high capacity (2 micrograms/mL). In cross-linking experiments, peak I appeared as two proteins of 165 and 174 kD; these mol wt were much higher than that of peak II-BP, previously estimated at 53,000. hGH complexed to peak II-BP remained fully immunoreactive with use of the anti-hGH antibodies of our assay. In plasma containing 10-20 micrograms/L hGH, the proportion of bound hormone (peak II) was 44.5 +/- 2.3%, whereas the amount of hGH in peak I was very low or undetectable. Specific binding of hGH to II-BP was lowest during the first year of life and highest in adulthood. No sex difference was found. I-BP is differentially regulated, since its binding activity was significantly lower in adults than in prepubertal children. Normal values for age should be taken into account to interpret GH-binding activity, particularly in children 2 yr of age or younger. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.