Toxins In Urine Research Articles

7Method for Detection of Naturally Occurring Toxins in Human Urine Using Liquid Chromatography High Resolution Mass Spectrometry.

Natural toxins present an ongoing risk for human exposure that requires a rapid, accurate diagnosis for proper response. In this study, a qualitative liquid chromatography high resolution mass spectrometry (LC-HRMS) method was developed and validated for the detection of a large, diverse selection of natural toxins. Data-dependent acquisition was performed to identify compounds with an in-house mass spectral library of 129 hazardous toxins that originate from plants, animals, and fungi. All 129 compounds were spiked into human urine, extracted, and evaluated for spectral library matching. Of these, 92 toxins met the quality criteria and underwent validation in urine matrix based on American National Standards Institute (ANSI) guidelines. A generalized workflow for method expansion was developed and enables the rapid addition of relevant compounds to the established method. This LC-HRMS method achieves efficient detection of natural toxins in urine, and the created workflow can rapidly increase compound coverage via method expansion.

Specific Alternation of Gut Microbiota and the Role of Ruminococcus gnavus in the Development of Diabetic Nephropathy.

In this study, we aim to investigate the precise alterations in the gut microbiota during the onset and advancement of diabetic nephropathy (DN) and examine the impact of Ruminococcus gnavus (R. gnavus) on DN. Eight-week-old male KK-Ay mice were administered antibiotic cocktails for a duration of two weeks, followed by oral administration of R. gnavus for an additional eight weeks. Our study revealed significant changes in the gut microbiota during both the initiation and progression of DN. Specifically, we observed a notable increase in the abundance of Clostridia at the class level, higher levels of Lachnospirales and Oscillospirales at the order level, and a marked decrease in Clostridia_UCG-014 in DN group. Additionally, there was a significant increase in the abundance of Lachnospiraceae, Oscillospiraceae, and Ruminococcaceae at the family level. Moreover, oral administration of R. gnavus effectively aggravated kidney pathology in DN mice, accompanied by elevated levels of urea nitrogen (UN), creatinine (Cr), and urine protein. Furthermore, R. gnavus administration resulted in down-regulation of tight junction proteins such as Claudin-1, Occludin, and ZO-1, as well as increased levels of uremic toxins in urine and serum samples. Additionally, our study demonstrated that orally administered R. gnavus up-regulated the expression of inflammatory factors, including nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) and Interleukin (IL)-6. These changes indicated the involvement of the gut-kidney axis in DN, and R. gnavus may worsen diabetic nephropathy by affecting uremic toxin levels and promoting inflammation in DN.

Late-onset schizophrenia: a differential diagnosis

IntroductionRegarding the diagnosis of schizophrenia, a peak of onset of symptoms is considered at 25 years. The debut after 60 years is considered late onset and is rare, generating controversies in the diagnosisObjectivesWe present the case of a 58-year-old patient with no personal or family history of mental health, who came to the emergency room for the first time, reporting feeling in danger. He comments itching on his skin, verbalizing seeing bugs running through it, relating this phenomenon to “witchcraft by my brothers”, he also refers to feeling like “they watch my thoughts and block it through a mobile application, they enter through my eye right and this gives me less vision and a headache. He also refers to having the ability to listen to how his brothers talk about how they are going to “hurt me.” Psychopathologically, we highlight that she is oriented in the three spheres, presenting delusional ideation with an experience of harm, a phenomenon of thought theft and auditory and tactile hallucinations.MethodsAnalytical and imaging tests, as well as toxins in urine, were negative.ResultsDiagnosis of psychotic episode is made to see evolution. The clinic partially yields to treatment with atypical antipsychotics. At this time, the patient has no awareness of the disease.ConclusionsDespite being a diagnosis that is scarcely prevalent, once organic disease has been ruled out.Disclosure of InterestNone Declared

S3241 A Case of Autoimmune Hepatitis in the Setting of the Janssen COVID-19 Vaccine

Introduction: Autoimmune hepatitis (AIH) is an inflammatory autoimmune condition that can result in chronic liver disease, it can be caused by the interaction of hereditary, epigenetic, immune, and environmental triggers. Vaccine related AIH is an uncommon phenomenon that has been reported in the presence of other vaccinations.. We present a unique case of AIH in the setting of the Janssen SARS-CoV-2 vaccine (JJ). Case Description/Methods: A 45-year-old female with a history ofHTN, DM, GERD, and obesity presented from her primary care physician’s office with abnormal liver chemistries in a hepatocellular pattern. Her hepatic profile showed AST 1119, ALT 988, AP 130, and total bilirubin of 1.5 (R-factor 22.8). Given the magnitude and rapidity of the elevated chemistries a broad serologic workup was initiated. She was negative for hepatitis A, B, and C, as well as HSV 1/2, EBV, CMV, HIV, VZV, and adenovirus. Her ferritin and iron studies did not suggest iron overload. Her ceruloplasmin and alpha 1 antitrypsin levels were within acceptable ranges. Her ANA was negative; her smooth muscle antibody was positive (1:320), IgG was elevated to 3,451 mg/dL. Her urine toxin screen was negative. She had chronically been on HCTZ and took occasionally seamoss supplements. She denied using tylenol or any other over the counter or herbal supplements. In the past month she had received a single dose of the Janssen SARS CoV-2 vaccination (Johnson and Johnson). Her liver chemistries prior were AST 17, ALT 14, AP 52, and total bilirubin of 0.3. She subsequently underwent a liver biopsy which showed moderate (grade 3) lobular inflammatory activity, mild (grade 2) periportal activity, and mild (stage 2) fibrosis with an abundance of plasma cells consistent with autoimmune hepatitis. Treatment was initiated with oral prednisone with a resulting improvement in her liver chemistries (Figure). Discussion: Autoimmune hepatitis in the setting of recent administration COVID 19 vaccine administration has been rarely reported. We present a unique case of COVID vaccine induced autoimmune hepatitis from a non-mRNA based vaccine. It has been suggested that possible mechanisms may include molecular mimicry or bystander activation of dormant autoreactive T-helper cells for both tissue-specific and non-tissue-specific reactions. We aim to shed light to this interesting paradigm and to spark the scientific discourse to further delineate potential mechanisms leading to this uncommon complication.Figure 1.: Liver biopsy which showed moderate (grade 3) lobular inflammatory activity, mild (grade 2) periportal activity, and mild (stage 2) fibrosis with an abundance of plasma cells consistent with autoimmune hepatitis.

Surface Shave: Revealing the Apical-Restricted Uroglycome.

This study aimed to investigate the highly differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification, and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal bladder urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans, the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabeling of the apically expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed for comparison with the N-glycome of the total porcine bladder urothelial cells and thus identification of apical surface glycans as candidates implicated in the urothelial barrier function. Data are available in MassIve: MSV000087851.

Simultaneous determination of 12 lipophilic shellfish toxins in plasma and urine by ultra-high performance liquid chromatography-tandem mass spectrometry

生物样品中脂溶性贝类毒素的检测,可为食物中毒等突发公共卫生事件的流行病学调查以及中毒者的临床救治提供技术支持。目前的研究存在目标化合物少,以及方法前处理复杂、灵敏度低等问题。该研究通过优化前处理和色谱分离技术,建立了超高效液相色谱-串联质谱法测定血浆、尿液中12种脂溶性贝类毒素的方法。实验对提取试剂以及流动相的选择进行了优化,采用乙腈对尿液和血浆样品进行提取。采用Phenomenex Kinetex C18色谱柱(50 mm×3 mm, 2.6 μm)进行分离,以0.05%(v/v)氨水水溶液、90%(v/v)乙腈水溶液为流动相,以流速0.40 mL/min梯度洗脱时,12种目标化合物分离效果最好。串联质谱的离子源为电喷雾离子(ESI)源,采用多反应监测(MRM)模式检测。12种目标物的基质效应均在0.8~1.1之间,表明该前处理方法的基质干扰低,采用外标法可对化合物进行准确定量。12种贝类毒素的线性范围为0.03~36.25 μg/L,相关系数均大于0.995。尿液检测的方法定量限为0.23~0.63 μg/L,血浆检测的方法定量限为0.31~0.84 μg/L。3个加标水平的回收率为72.7%~124.1%,日内精密度为2.1%~20.0%,日间精密度为2.1%~15.3%。利用该方法检测健康人尿液和血浆样本,以及经腹腔注射12种贝类毒素的小鼠尿液和血液样本。20份健康人样本中未检出目标物,20份小鼠样本中12种贝类毒素均有检出。该方法操作简便,样品取样量少,方法灵敏高,适用于血浆和尿液中脂溶性贝类毒素的快速检测。

Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry

鹅膏肽类毒素是一类环状多肽类蘑菇毒素,中毒后会造成急性肝损伤,病死率非常高。我国因误食野生毒蘑菇导致的中毒事件常有发生,测定人尿中鹅膏肽类毒素的浓度,可为临床早期诊断和救治提供有价值的信息。该研究建立了TurboFlow (TF)在线净化-液相色谱-三重四极杆质谱快速定量检测尿液中5种鹅膏肽类毒素(α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、羧基二羟鬼笔毒肽和二羟鬼笔毒肽)的新方法。尿液样品经高速离心后,直接注入TurboFlow在线净化-液相色谱-串联质谱进行分析。对影响TF在线净化的参数如TF净化柱、上样溶剂、洗脱溶剂、转移流速、转移时间等进行了优化。在优化后的实验条件下,以TurboFlowTMCyclone柱(50 mm×0.5 mm)为净化柱,Hypersil GOLD C18柱(100 mm×2.1 mm)为分析柱,甲醇和4 mmol/L乙酸铵为流动相进行梯度洗脱,电喷雾正离子选择反应监测(SRM)模式下进行检测,基质匹配外标法定量。结果表明,鹅膏肽类毒素在1.0~50.0 μg/L范围内呈现良好的线性关系,相关系数均可达到0.997以上。方法的检出限为0.15~0.3 μg/L,定量限为0.5~1.0 μg/L。在2.0、5.0和10.0 μg/L的加标水平下,5种鹅膏肽类毒素的日内和日间回收率分别为87.0%~108.6%和86.8%~112.7%,日内、日间相对标准偏差(RSD)均小于14.5%。该检测方法准确、快速、灵敏度高、易操作,适用于公共卫生应急检测或临床中毒病因识别检测。

Urinary essential elements of young children with autism spectrum disorder and their mothers

BackgroundEven though the cause of autism spectrum disorders (ASD) remains unknown, the current understanding points towards complex interactions between environmental and genetic factors. One important environmental factor to consider is intake of toxic and essential elements, and their role in metabolism. Essential elements have received considerably less attention in the literature than the presence of toxins in urine. MethodThe purpose of this investigation is to comprehensively assess the association between urinary element compositions of 28 mothers who had young children with ASD and 29 mothers who had young typically developing (TD) children, and in a subset of their children (21 with ASD and 26 TD). ResultsThe results show that there are significant differences between the ASD and TD children cohorts’ concentrations for four specific elements (sulfur, phosphorous, molybdenum, and tin). Utilizing multivariate statistical techniques (Fisher’s discriminant analysis and support vector machines), it was possible to distinguish the ASD from the TD children groups with an 81 % accuracy after cross-validation utilizing the four significantly different elements. However, among the mother cohorts assessed, there were no significant differences between those that had children with ASD and those with TD children. There was a significant correlation of levels of phosphorus and sulfur in the children with ASD (r = 0.63, p = 3.0E-3) and in the TD children (r = 0.47, p = 0.02). ConclusionsNotable differences were observed between the elemental concentration in urine of children with ASD and their TD peers. Analyzing cellular pathways related to these elements are promising areas of future research.

Comparison of clinical effect of different dialysis frequency and methods in the treatment of chronic renal failure uremic patients

Objective To observe the clinical efficacy of different dialysis frequency and methods in the treatment of chronic renal failure uremia patients, and to provide a reliable reference for clinical practice. Methods From February 2016 to February 2017, 140 chronic renal failure uremic patients who admitted to the Department of Nephrology at Taizhou Central Hospital were selected and divided into the observation group and the control group by complete random distribution method, with 70 cases in each group.The patients in the control group received 3 times of regular hemodialysis treatment per week, while the observation group received 3 times of regular hemodialysis combined with 1 time of hemodiafiltration treatment per week.The clinical treatment effects and complications of the two groups were compared. Results Compared with blood uric acid(BUA)[(532.09±71.05)mmol/L], serum creatinine(Scr)[(734.34±63.08)μmol/L], blood urea(BUN)[(23.84±2.58)mmol/L], serum phosphorus[(1.98±0.37)mmol/L], parathyroid hormone(PTH)[(464.37±113.92)ng/L], β2 microglobulin(β2-MG)[(13.48±6.38)mg/L]in the control group, BUA[(495.89±58.23)mmol/L], Scr[(684.34±77.32)μmol/L], BUN[(20.62±1.77)mmol/L], serum phosphorus[(1.74±0.44)mmol/L], PTH[(352.14±123.25)ng/L], β2-MG[(9.58±5.45)mg/L] were significantly lower in the observation group, the differences were statistically significant (t=3.29, 4.19, 8.61, 3.49, 5.59, 3.88, all P 0.05). Conclusion Hemodialysis 3 times combined with 1 time of hemodiafiltration per week has significant advantage in the treatment of chronic renal failure uremia patients, which can effectively remove urine toxin molecules and reduce the incidence of complications, and it is worthy of clinical application. Key words: Renal failure, chronic; Uremia; Hemodialysis; Hemodiafiltration; Comparative effectiveness research

First insights into Alternaria multi-toxin in vivo metabolism

Alternaria mycotoxins frequently contaminate agricultural crops and may impact animal and human health. However, data on mammalian metabolism and potential biomarkers of exposure for human biomonitoring (HBM) are scarce. Here, we report the preliminary investigation with respect to metabolism and excretion of Alternaria toxins in Sprague Dawley rats. Four animals were housed in metabolic cages for 24 h after gavage administration of an Alternaria alternata culture extract containing ten known toxins. LC–MS/MS analysis of 17 Alternaria toxins in urine and fecal samples allowed to gain first insights regarding xenobiotic metabolism and excretion rates. Alternariol (6–10%), alternariol monomethyl ether (AME, 6–7%) and tenuazonic acid (up to 55%) were recovered in urine and fecal samples (9%, 87%, 0.3%, respectively), while perylene quinones administered at comparatively high levels, were either determined at very low levels (up to 0.5% altertoxin I in urine and 15% in feces; 0.2% alterperylenol in urine and 3% in feces) or not at all (altertoxin II, stemphyltoxin III). AME-3-sulfate, which was not present in the administered extract, was determined in urine, representing up to 23% of the AME intake. Critical evaluation of the applied sample preparation protocol and LC–MS/MS analysis revealed interesting preliminary results and information crucial for improving follow-up experiments.

Toxic Action Reevaluation of Okadaic Acid, Dinophysistoxin-1 and Dinophysistoxin-2: Toxicity Equivalency Factors Based on the Oral Toxicity Study

Background/Aims: Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic shellfish poisoning (DSP) in humans due to ingestion of contaminated shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg^-1 shellfish meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs). Methods: Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay. Results: Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD₅₀) was 487 µg kg^-1 bw for DTX1, 760 µg kg^-1 bw for OA and 2262 µg kg^-1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3. Conclusion: This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated.

Fast and quantitative determination of saxitoxin and neosaxitoxin in urine by ultra performance liquid chromatography-triple quadrupole mass spectrometry based on the cleanup of solid phase extraction with hydrophilic interaction mechanism.

Saxitoxin (STX) and neosaxitoxin (NEO) are water-soluble toxins and their cleanup in bio-matrix is a hot topic but difficult problem. A fast and quantitative determination method for STX and NEO in urine was developed using ultra performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) based on the cleanup of solid phase extraction (SPE) with hydrophilic interaction (HILIC) mechanism. Acetonitrile/methanol/water mixture was used to extract the toxins in urine. Polyamide (PA) was used as HILIC SPE material to clean the toxins in sample matrix. The limits of detection were 0.2ngmL-1 for STX and 1ngmL-1 for NEO in urine. The linear ranges were 0.5ngmL-1-99.2ngmL-1 with the correlation coefficient of r=0.9992 for STX and 2.1ngmL-1-207ngmL-1 with r=0.997 for NEO in urine matrix. The recoveries at three spiking levels were 81.5%-117% with the relative standard deviations (RSDs) of 5.4%-8.5% for STX and 89.0%-118% with the RSDs of 6.7%-9.1% for NEO. STX was found in all the 6 patients' urines while NEO was only found in one sample from an intoxication case.

Simultaneous determination of zearalenone, deoxynivalenol and their metabolites in bovine urine as biomarkers of exposure

A feeding trial with 30 dairy cows which were fed rations with three different concentrations of zearalenone (ZEA) and deoxynivalenol (DON) contaminated maize was carried out to examine the ZEA and DON concentration in urine. German Holstein cows (n=30) were divided into three groups (n=10 in each) which received diets with following toxin concentrations: CON (0.02 mg ZEA and 0.07 mg DON, per kg dry matter (DM)), FUS-50 (0.33 mg ZEA and 2.62 mg DON, per kg DM), FUS-100 (0.66 mg ZEA and 5.24 mg DON, per kg DM). For urine analysis, a reliable, cost-efficient and sensitive method for simultaneous determination of ZEA, DON and their metabolites was developed. The method comprises a solid phase extraction clean-up on Oasis HLB cartridges followed by LC-MS/MS measurement. ZEA, α-zearalenol, β-zearalenol, DON and de-epoxydeoxynivalenol (DOM) could be detected in the urine samples of the feeding trial. Thereby, DON was almost completely metabolised to DOM (83-98%) independent of the DON exposure. Moreover, conjugated toxins were the major urinary metabolites based on results of the analysis with β-glucuronidase treated and untreated samples. Furthermore, relationships between toxin intake and urinary toxin concentration could be established. In conclusion, increased urine toxin concentrations may hint on toxin exposure through the diets and thus the mycotoxins ZEA and DON and their detected metabolites could be used as biomarkers of exposure.

Conversion disorder: Not a malingering matter

A 12-year-old girl was seen by her family physician with a chief complaint of seizures. Over the past several weeks, she has suffered from attacks of bilateral arm jerking, followed by bilateral leg jerking. Often, her head shakes violently from side to side and her eyes are seen to roll back in her head. These spells are not accompanied by bladder or bowel incontinence, but often the patient bites her tongue and kicks over tables or strikes family members during an episode. These incidents typically follow episodes of emotional outburst and can wax and wane for 20 min to 30 min with varying intensity. She has no significant medical history and takes no medications. Her school performance is excellent. She is a competitive gymnast who spends 15 h per week training. On examination, her vitals signs are normal, without orthostatic hypotension, and her neurological evaluation is unremarkable. Medical investigations including glucose, electrolytes, a urine toxin screen and an EEG are all within normal limits.

Direct quantification of deoxynivalenol glucuronide in human urine as biomarker of exposure to the Fusarium mycotoxin deoxynivalenol

The direct quantification of deoxynivalenol glucuronide (DON-GlcA) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application as a biomarker of exposure to the Fusarium mycotoxin deoxynivalenol (DON) is reported. Usually, DON exposure is estimated from dietary average intakes or by measurement of the native toxin in urine after enzymatic hydrolysis with β-glucuronidase. These methods are time-consuming, expensive, and fail to determine the ratio of DON to DON-GlcA in a simple one-step procedure. One of the main reasons for the use of indirect methods is the unavailability of DON-GlcA standards. Consequently, DON-3-O-glucuronide (D3GlcA) was synthesized and used to develop a method allowing quantification of both DON and D3GlcA by a simple "dilute and shoot" approach without the need for any cleanup. Limit of detection and apparent recovery of D3GlcA was 3 μg l(-1) and 88%, respectively. The identity of D3GlcA in human urine was confirmed by comparison with LC-MS/MS measurements of the synthetically produced D3GlcA standard which was also used for external calibration. The applicability of the method was demonstrated through the analysis of urine samples obtained from a volunteer during regular and cereal-restricted diet, respectively. In regular-diet urine samples, D3GlcA was quantified in concentrations >30 μg l(-1) by this approach.

Critical Conversations

VignetteA 36-year-old homeless man was brought in to the emergency room with altered mental status and was unable to give a history. Based on prior emergency visits, he has a history of IV drug abuse and alcohol abuse. His vital signs on arrival are the following: HR of 110 (regular), BP of 102/58, temperature of 98.0, and respiratory rate of 8/min. His Glasgow Coma Scale score is 7. Physical examination results are that he is grossly obtunded, responds only to deep painful stimuli, is nonverbal, and moves all extremities. Pupils are small, 2 to 3 mm, and reactive. His heart reveals regular tachycardia. Lungs are clear to auscultation. Abdomen is within normal limits. His extremities are warm to touch. Skin reveals ecchymosis. His initial head CT finding is negative for bleeding, infarct, or mass. He has severe metabolic acidosis, and the urine toxin screen result is positive for cocaine and opiates. His creatinine kinase enzyme is greater than 20,000 IU/L. Peripheral venous access attempts are unsuccessful.The right internal jugular vein is accessed via a needle with ultrasound guidance, but a guidewire failed to pass on multiple attempts. The emergency physician inserts a right femoral line triple lumen catheter without ultrasound. The patient is intubated via an endotracheal tube for airway protection and admitted to the medical ICU.

Síndrome opsoclono-mioclono-atáxico paraneoplásico

Opsoclonus myoclonus ataxia syndrome (OMAS) is a very infrequent paraneoplastic or postinfectious movement disorder, which may occur at any age, most commonly between 6 and 36 months of age. In four days, a previously healthy 30-month-old girl progressively developed gait instability, intention tremor, dysarthric speech, irritability and altered sleep. Physical and neurological examination did not reveal additional deficits. She had had a transient exanthema without fever three weeks before. Basic blood analysis, serologies, cultures, urine toxin detection, EEG and cerebral CT were normal. Lumbar puncture showed minimal lymphocytosis. On the fifth day following the onset of symptoms, the ataxia worsened, precluding sitting, and the tremor was aggravated by intentional myoclonus. Chaotic saccadic, large amplitude multidirectional but conjugated eye movements appeared. An opsoclonus (OMAS) was suspected and a chest X-ray and CT revealed a paravertebral thoracic mass. Surgery confirmed a localized ganglioneuroblastoma. Blood neuron-specific enolase and urine catecholamine levels were normal. Opsoclonus disappeared with high doses of prednisone and following surgery. Ataxia improved but the patient still required low daily doses of steroids for one year. Conclusions. The diagnosis of OMAS is based on clinical findings. Opsoclonus is a specific clinical sign that all clinicians must recognize. The presence of opsoclonus should always prompt the search for a neural-crest tumour, as one of the most important precipitating causes. The prognosis of this tumour is usually excellent, but neurological symptoms frequently relapse due to the tapering of steroids.

Prevalence of zeranol, taleranol and Fusarium spp. toxins in urine: implications for the control of zeranol abuse in the European Union

There is currently little information concerning the prevalence of zeranol and taleranol in animal urine following metabolism of the naturally occurring Fusarium spp. toxins. An epidemiological study is described which involves four European Union control laboratories in which 8008 urine samples were screened for the presence of zeranol using a time-resolved fluoroimmunoassay (TR-FIA). Of these samples, 93.6% screened negative for zeranol. All samples testing positive for zeranol were then analysed with a confirmatory method. Based on the confirmatory results, the TR-FIA-positive samples were then categorized as false-positive, true-positive or ‘equivocal’ (zeranol/taleranol and the Fusarium spp. toxins detected). The true-positive samples represented only 0.05% of the total number of samples (n = 4). After statistical analysis, 170 of 174 equivocal samples proved to belong to a ‘normal’ population in which the amount of zeranol/taleranol could be related to the total amount of Fusarium spp. toxins through a linear regression with a 99% prediction interval. This suggested that the presence of zeranol in these samples might be due to in vivo metabolism of the Fusarium spp. toxins. The presence of zeranol in the four remaining ‘outliers’ might be attributable to zeranol abuse rather than to natural contamination. The results are of interest for control laboratories as they might provide an analytical tool to help distinguish between abuse and natural contamination in zeranol testing.

Quantitative variability in the biodistribution and in toxinokinetic studies of the three main alpha toxins from the Androctonus australis hector scorpion venom

Scorpion stings represent a medical problem in numerous countries. The scorpion Androctonus australis hector produces three alpha toxins (Aah I to III), which are responsible for most of the lethality in mammals. These toxins act on sodium channel and do not cross-react immunologically. We used RIA and ELISA to measure the concentrations of these three toxins in plasma, urine and different organs after i.v. and s.c. injections of water extracts of venoms in rabbits or mice. In both animals, the toxins rapidly appeared in plasma after s.c. injection as it was previously described for the whole venom. However, the toxins disappeared from the blood more quickly than did other main components of the venom. Thus, serotherapy must be initiated immediately to prevent the toxin from reaching its target. We also detected the toxins in urine, kidneys, heart and lungs, but not in the brain. However, the concentration of Aah II was always lower than that of Aah I. Analysis of five samples of venom collected in different areas of southern Tunisia showed that a large polymorphism exists for the three toxins. This is yet another difficulty for serotherapy as there is no cross-antigenicity between them.

Development and validation of dry reagent time-resolved fluoroimmunoassays for zeranol and α -zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml-1) demonstrating that up to 150 ng ml-1 of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 96/23/EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin α-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml-1). Only the addition of diluted sample extract is required to perform these dry-reagent TRFIAs, the results being available within 1h of extract application. The EU-funded project ‘Natural Zeranol’ (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.