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Background: The impact of peanut allergy is large and accidental ingestion of peanut can lead to severe reactions. Currently used diagnostic tests, such as skin prick tests (SPT) and determination of specific immunoglobulins (IgE) have, however, limited sensitivity and specificity. Therefore, new tools have to be developed to improve the accuracy of the diagnostic work-up of food-allergic patients. Comprehensive metabolite analysis may provide biomarkers for diagnosing food allergy as metabolite levels reflect actual physiological conditions. We investigated whether metabolites can be found that discriminate between peanut-allergic patients and non-peanut-allergic subjects. Such metabolites may be used for future diagnostic purposes. Methods: Plasma and saliva samples were obtained from 23 participants (12 peanut allergic and 11 peanut tolerant) prior to and after a peanut challenge and measured with 1H nuclear magnetic resonance (NMR) spectroscopy with subsequent multivariate data analysis. Results: Clear differences were observed between NMR spectra of peanut-allergic and peanut-tolerant subjects in plasma as well as saliva. Allergic patients already showed aberrant metabolite levels prior to peanut ingestion, thus before the onset of allergic reactions. Conclusion: This pilot study shows that aberrant metabolite levels as determined by NMR in combination with multivariate statistics may serve as novel biomarkers for food allergy.

1H nuclear magnetic resonance (NMR) spectroscopy of rat urine in combination with pattern recognition analysis was evaluated for early noninvasive detection of toxicity of investigational chemical entities. Bromobenzene (B) and paracetamol (P) were administered at five single oral dosages between 2 and 500 mg/kg and between 6 and 1800 mg/kg, respectively. The sensitivity of the proposed method to detect changes in the NMR spectra 24 and 48 h after single dosing was compared with histopathology and biochemical parameters in plasma and urine. Both B and P applied at the highest dosages induced liver necrosis and markedly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) plasma levels. At dosages of 125 mg/kg B and 450 mg/kg P, liver necrosis and changes in AST and ALT were less pronounced, while at lower dose levels these effects could not be detected. Changes in kidney pathology or standard urine biochemistry were not observed at any of these dosages. Evaluation of the total NMR dataset showed 80 signals to be sensitive for B and P dosing. Principal component analysis on the reduced dataset revealed that NMR spectra were significantly different at dosages above 8 mg/kg (B) and 110 mg/kg (P) at both sampling times. This implies a 4- to 16-fold increased sensitivity of NMR versus histopathology and clinical chemistry in recognizing early events of liver toxicity. © The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

The introduction of the concept of systems biology, enabling the study of living systems from a holistic perspective based on the profiling of a multitude of biochemical components, opens up a unique and novel opportunity to reinvestigate natural products. In the study of their bioactivity, the necessary reductionistic approach on single active components has been successful in the discovery of new medicines, but at the same time the synergetic effects of components were lost. Systems biology, and especially metabolomics, is the ultimate phenotyping. It opens up the possibility of studying the effect of complex mixtures, such as those used in Traditional Chinese Medicine, in complex biological systems; abridging it with molecular pharmacology. This approach is considered to have the potential to revolutionize natural product research and to advance the development of scientific based herbal medicine. Copyright © 2005 John Wiley & Sons, Ltd.

Ginkgo biloba extract has been advocated for the improvement of blood circulation in circulatory disorders. This study investigated the effect of the Gingko biloba extract EGb 761 on skin blood flow in healthy volunteers and accompanying changes in urinary metabolites. Twenty-seven healthy middle-aged subjects participated in a randomized, double-blind, placebo-controlled, crossover study. Subjects received 240 mg/d EGb 761 or placebo for periods of 3 weeks. Skin blood flow was measured on the forefoot using laser Doppler flowmetry and changes in urinary metabolites were identified by a combination of nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis (MVDA). These measurements were performed on 24-h urine samples collected at the end of the intervention periods. Following EGb 761 treatment, overall mean skin blood flow was significantly reduced as compared with placebo. Remarkably, the change of skin blood flow after EGb 761 intervention was proportionally related to blood flow after placebo treatment: subjects showed either an increased, decreased or unaltered skin blood flow. NMR/MDVA analyses showed that urinary metabolic patterns differed depending on the change in baseline blood flow after treatment with EGb 761. The present findings substantiate that EGb 761 has a multi-directional modulating action on blood flow in healthy subjects and support findings of a vasoregulatory role of this extract. Moreover, the results indicate that metabolic fingerprinting provides a powerful means to identify biochemical markers that are associated with functional changes.

Osteoarthritis (OA), one of the most common diseases among the elderly, is characterized by the progressive destruction of joint tissues. Its etiology is largely unclear and no effective disease-modifying treatment is currently available. Metabolic fingerprinting provides a novel tool for the identification of biomarkers. A metabolic fingerprint consists of a typical combination of metabolites in a biological fluid and is identified by a combination of (1)H NMR spectroscopy and multivariate data analysis (MVDA). The current feasibility study was aimed at identifying a metabolic fingerprint for OA and applying this in a nutritional intervention study. Urine samples were collected from osteoarthritic male Hartley guinea pigs (n = 44) at 10 and 12 mo of age, treated from 4 mo onward with variable vitamin C doses (2.5-3, 30 and 150 mg/d) and from healthy male Strain 13 guinea pigs (n = 8) at 12 mo of age, treated with 30 mg vitamin C/d. NMR measurements were performed on all urine samples. Subsequently, MVDA was carried out on the data obtained using NMR. An NMR fingerprint was identified that reflected the osteoarthritic changes in guinea pigs. The metabolites that comprised the fingerprint indicate that energy and purine metabolism are of major importance in OA. Metabolic fingerprinting also allowed detection of differences in OA-specific metabolites induced by different dietary vitamin C intakes. This study demonstrates the feasibility of metabolic fingerprinting to identify disease-specific profiles of urinary metabolites. NMR fingerprinting is a promising means of identifying new disease markers and of gaining fresh insights into the pathophysiology of disease.

Metabolic fingerprints are novel measurement tools to evaluate the biochemical status of a living organism by using 1H NMR and multivariate data analysis (MVDA). In this way, a quick evaluation of changes in health or diseased state can be made, reflected in alterations of metabolic patterns. Normally, metabolic finger-printing is based on in vivo studies. These studies often represent a labor-intensive and expensive manner of investigation. In vitro studies are not hampered by these disadvantages, thus constituting an interesting alternative. In this research, results are presented of a pilot experiment in which metabolic fingerprinting was combined with an in vitro model. For this purpose, differentiated Caco-2 cells were exposed to inulin and its fermentative metabolites, both dissolved in culture medium. Cells were incubated for 0 or 48 h. Cell fractions were analyzed by NMR, then subsequently with MVDA. Differences in treatment provided detectable variations in the time of metabolic patterns of cell contents. Results indicated that glucose metabolism linked to glutamate was of major importance in the effects of inulin and its metabolites on Caco-2 cells under the conditions of our study. Metabolic fingerprinting in combination with an in vitro model appears to be a feasible technique with which to visualize metabolic patterns of cell contents and provides an efficient place for the generation of hypotheses about the metabolic pathways involved. In vitro metabolic fingerprinting may be of great benefit in the future for a better understanding of the relationship between nutrition and health.

Estrogen-induced pituitary lesions in rats were studied in time-sequence experiments using magnetic resonance imaging (MRI), hormone determinations and light microscopy. The main purpose of the study was to evaluate the usefulness of MRI in comparison with conventional biochemical and histopathological methods for detecting the pituitary lesions as early as possible and to follow their development. Measurements were made at 15 time points, ranging from 1 h to 272 days after s.c. implantation of the estrogen pellet. High-resolution T1 weighted sagittal images with 2 mm slice thickness were made with a 2 Tesla 30 cm small-bore MRI system. Radioimmunoassay (RIA) was used to determine the different pituitary hormones. Convential histopathology and immunoperoxidase staining methods were used to characterize the pituitary lesions and visualize the hormone-producing pituitary cells respectively. The first histopathological pituitary changes (enlarged acidophilic cells with increased number of vacuoles) were seen at day 2 after initiation of the estrogen treatment, while at day 4 the first immunohistochemical changes (increased number of prolactin-positive cells) were encountered. Significantly increased prolactin levels in blood plasma occurred from day 9 onwards. Also at day 9, changes of the pituitary gland were first visible on MR images, showing rounding of the anterior edge of the gland. Gradual enlargement of the pituitary caused by hyperplasia of hypertrophic prolactin-positive cells could be followed by MRI, and later on pituitary tumors were recognized, their images being heterogeneous due to great differences in signal intensity ranging from hypo- or iso- to hyperintense. Signal intensities of hemorrhagic tumor areas varies widely due to variation in the blood flow maintained in these areas. It was concluded that MRI is a powerful tool for detecting enlargement and tumors of the pituitary gland in rats. This method allows the development of such lesions to be followed in one and the same animal, thereby reducing the need of interim kills and thus the number of animals to be used. Chemicals/CAS: prolactin, 12585-34-1, 50647-00-2, 9002-62-4; Estradiol, 50-28-2; Estrogens; Pituitary Hormones

A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition is proposed for early detection of toxicity of investigational compounds in rats. The method is applied to detect toxicity upon administration of 13 toxic reference compounds and one nontoxic control compound (mianserine) in rats. The toxic compounds are expected to induce necrosis (bromobenzene, paracetamol, carbon tetrachloride, iproniazid, isoniazid, thioacetamide), cholestasis (α-naphthylisothiocyanate (ANIT), chlorpromazine, ethinylestradiol, methyltestosterone, ibuprofen), or steatosis (phenobarbital, tetracycline). Animals were treated daily for 2 or 4 days except for paracetamol and bromobenzene (1 and 2 days) and carbon tetrachloride (1 day only). Urine was collected 24 h after the first and second treatment. The animals were sacrificed 24 h after the last treatment, and NMR data were compared with liver histopathology as well as blood and urine biochemistry. Pathology and biochemistry showed marked toxicity in the liver at high doses of bromobenzene, paracetamol, carbon tetrachloride, ANIT, and ibuprofen. Thioacetamide and chlorpromazine showed less extensive changes, while the influences of iproniazid, isoniazid, phenobarbital, ethinylestradiol, and tetracycline on the toxic parameters were marginal or for methyltestosterone and mianserine negligible. NMR spectroscopy revealed significant changes upon dosing in 88 NMR biomarker signals preselected with the Procrustus Rotation method on principal component discriminant analysis (PCDA) plots. Further evaluation of the specific changes led to the identification of biomarker patterns for the specific types of liver toxicity. Comparison of our rat NMR PCDA data with histopathological changes reported in humans and/or rats suggests that rat NMR urinalysis can be used to predict hepatotoxicity. © The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

This study investigated whether integrated analysis of transcriptomics and metabolomics data increased the sensitivity of detection and provided new insight in the mechanisms of hepatotoxicity. Metabolite levels in plasma or urine were analyzed in relation to changes in hepatic gene expression in rats that received bromobenzene to induce acute hepatic centrilobular necrosis. Bromobenzene-induced lesions were only observed after treatment with the highest of 3 dose levels. Multivariate statistical analysis showed that metabolite profiles of blood plasma were largely different from controls when the rats were treated with bromobenzene, also at doses that did not elicit histopathological changes. Changes in levels of genes and metabolites were related to the degree of necrosis, providing putative novel markers of hepatotoxicity. Levels of endogenous metabolites like alanine, lactate, tyrosine and dimethylglycine differed in plasma from treated and control rats. The metabolite profiles of urine were found to be reflective of the exposure levels. This integrated analysis of hepatic transcriptomics and plasma metabolomics was able to more sensitively detect changes related to hepatotoxicity and discover novel markers. The relation between gene expression and metabolite levels was explored and additional insight in the role of various biological pathways in bromobenzene-induced hepatic necrosis was obtained, including the involvement of apoptosis and changes in glycolysis and amino acid metabolism. The complete Table 2 is available as a supplemental file online at http://taylorandfrancis.metapress.com/openurlasp?genre=journal&issn=0192-6233. To access the file, click on the issue link for 33(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org. Copyright © by the Society of Toxicologic Pathology.

Pituitary hypertrophy and tumors were induced in male Sprague Dawley rats using estradiol-17 beta. This tumor model generates a variety of pituitary lesions which are relevant to human pituitary disease. In order to characterize these lesions, gadolinium DTPA was injected intravenously into the tail vein of estrogen treated and control rats. High resolution T1-weighted MR images, pre- and postenhancement, were obtained at 8 different time points spanning 300 days following the subcutaneous implantation of the estrogen pellets. Images with 2-mm slice thickness were made with a 2 Telsa small-bore MR imaging system. Both normal and tumorous pituitaries were found to enhance with contrast agent, but contrast uptake was not uniform. Gd-DTPA distribution was sensitive to the different types of lesions generated in the course of this study. Digital subtraction of congruent images, pre- and postcontrast, provided difference images reflecting contrast concentration and allowed identification of subtle enhancement effects. Hypertrophic pituitaries displayed uptake of contrast, but the distribution of contrast agent was nonuniform and appeared mottled. A bright rim enhancement was often seen anterior to the pituitary gland, most likely arising from the oculomotor nerves and arachnoid. Histological slices in the same anatomical plane as the MR images were obtained on the animals allowing identification of individual lesions. Cystic areas within tumors were found to give strong contrast enhancement in less than five min postinjection. Solid and hemorrhagic areas of the pituitary tumor were hypo- to isointense relative to surrounding brain and did not take up contrast agent. Significant perfusion in these areas apparently does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen-induced pituitary hypertrophy has been studied using magnetic resonance imaging in a group of 15 control and 30 experimental rats. Following the subcutaneous implantation of an estrogen pellet, differences in the anatomical appearance of the pituitary gland of the implanted rats can be detected in as little as 16 days, when compared to the control animals. The gland in the experimental animals appears diffusely enlarged with rounded margins, when viewed in sagittal T1-weighted magnetic resonance images. Additionally, a uniform signal intensity is not detected in the hypertrophic pituitary glands of estrogen-implanted rats, while in the control rats this is a common finding. A satellite study of 300 animals (treated in a manner identical to that in the imaging study) demonstrates that the weights of excised pituitaries in estrogen-treated rats increases to a statistically significant level in the interval 18 to 35 days. Changes in the appearance and volume of the pituitary gland observed with magnetic resonance imaging seem to have promise for the early detection of pituitary lesions in rats.