Rapid HPLC screening method for contaminants found in implicated L-tryptophan associated with eosinophilia myalgia syndrome and adulterated rapeseed oil associated with toxic oil syndrome.

In 1981 a massive food-borne epidemic, termed the toxic oil syndrome (TOS), occurred in Spain. Eight years later a closely related disease, the eosinophilia myalgia syndrome (EMS), was reported in the USA with many additional cases being reported worldwide. Although EMS was linked to the ingestion of contaminated L-tryptophan and TOS to aniline denatured rapeseed oil, the etiological agent(s) responsible for both diseases remains unknown. However, contaminants in both the oil and the dietary supplement are believed to have triggered these diseases, and there has been much speculation that a common contaminant may have caused both epidemics. In this report, methods for the facile preparation and HPLC analysis of EMS-implicated L-tryptophan and adulterated rapeseed oil samples associated with TOS are described which allow a direct comparison between the contaminants of both foodstuffs. A combination of solvent and solid phase extraction methods are demonstrated along with the application of C18 reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with on-line UV and MS detection. These methods have allowed us to determine for the first time, based upon this work, that there are no detectable common contaminants that possess a UV response, between EMS implicated L-tryptophan and TOS implicated rapeseed oil samples.

Structural characterization of contaminants found in commercial preparations of melatonin: similarities to case-related compounds from L-tryptophan associated with eosinophilia-myalgia syndrome.

On-line HPLC/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) in conjunction with NMR has been successfully employed to identify and structurally characterize seven contaminants found in three different commercial preparations of melatonin. Six of these contaminants were identified as analogues of impurities found in contaminated L-tryptophan (an over-the-counter dietary supplement) associated with the eosinophilia-myalgia syndrome (EMS) epidemic that occurred in the United States during 1989. In particular, our studies identified two compounds with MH+ = 249 to be hydroxymelatonin isomers. Four other compounds with MH+ = 477 were identified as melatonin-formaldehyde condensation products. These compounds are structural analogues of L-tryptophan contaminants, namely, 'peak C' and 'peak E' that were previously implicated as etiological agents causing EMS. It has been reported that melatonin consumption has resulted in eosinophilia in some humans taking high doses of this supplement. Although there has not been a major outbreak of EMS-like symptoms from consumption of melatonin, this study clearly suggests that tighter control and regulation of nutritional supplements sold and used as drugs is necessary.

Differential effects of inhibition of isoforms of cyclooxygenase (COX-1, COX-2) in chronic inflammation.

OBJECTIVE AND DESIGN: The anti-inflammatory effects of therapeutic dosing of drugs with greater selectivity for the inhibition of the constitutive (COX-1) or inducible isoform (COX-2) of cyclooxygenase were assessed in a model of chronic inflammation. METHODS: The murine chronic granulomatous tissue air pouch model involves the subcutaneous injection of air into the dorsum of mice followed 24 h later by the intrapouch injection of an inflammatory stimulus (0.5 ml of Freund's complete adjuvant containing 0.1% croton oil). Aspirin, more selective in vitro for the inhibition of COX-1 (10,200 (mg/kg) and nimesulide, a selective in vitro inhibitor of COX-2 (0.5, 5 mg/kg) were dosed p.o. daily from 3 days after injection of the inflammatory stimulus. Granuloma dry weight, vascularity and COX activity (measured as PGE2) were assessed at various time points throughout the inflammatory lesion to resolution at day 28. A second COX-2 inhibitor, NS 398 (0.1, 1, 10 mg/kg), was dosed p.o. daily from 3 days after the injection of the inflammatory stimulus and its effects on granuloma dry weight, vascularity and COX activity were measured at 7 days. RESULTS: Aspirin (200 mg/kg) significantly inhibited levels of PGE2 throughout the time course and at the lower dose (10 mg/kg) from day 14. Nimesulide (5 mg/kg) however, significantly increased levels of PGE2 at days 5 and 21, but at 0.5 mg/kg was without effect. Aspirin (200 mg/kg) significantly reduced granuloma dry weight at day 14 but had no effect on granuloma vascularity at day 7. In contrast, nimesulide (5 mg/kg) significantly increased granuloma vascularity at day 7 and granuloma dry weight at day 14. NS-398 at all doses had no effect on granuloma dry weight, vascularity or COX activity 7 days after the injection of the inflammatory stimulus. CONCLUSION: In this model of chronic inflammation, aspirin, more selective for the inhibition of COX-1 is more effective than the selective COX-2 inhibitors nimesulide and NS-398 at inhibiting granuloma dry weight, vascularity and COX activity.

Evaluation of the efficacy and safety of amethocaine gel applied topically before venous cannulation in adults.

We have assessed in a placebo-controlled, double-blind trial, the efficacy and safety of an amethocaine gel preparation for alleviating the pain of venous cannulation. Forty-two unpremedicated adult patients were allocated randomly to receive either amethocaine gel 1 g (4% w/w) or placebo gel 1 g applied to the dorsum of the hand for 40 min before venous cannulation. After removal of the gel, a 20-gauge cannula was inserted and the pain of cannulation assessed by a three-point rank score and a 100-mm visual analogue scale: 90% of patients who received amethocaine reported clinically acceptable topical anaesthesia. Both verbal rating scores and visual analogue scores for pain were significantly less with amethocaine. Slight erythema associated with amethocaine occurred in one patient and slight itching in two patients. No other adverse features were evident.

Comparison of percutaneous anaesthesia for venous cannulation after topical application of either amethocaine or EMLA cream.

We have compared, in a double-blind study, the efficacy of topical amethocaine cream 1 g (5% w/w) in alleviating the pain of venous cannulation with that of 5% EMLA cream 2.5 g. One hundred and twenty unpremedicated female patients undergoing minor gynaecological surgery, were allocated randomly to one of four groups: 5% EMLA cream 2.5 g for 30 min: 5% EMLA cream 2.5 g for 60 min; amethocaine cream 1 g (5% w/w) for 30 min; amethocaine cream 1 g (5% w/w) for 60 min. After removal of the cream, venous cannulation was performed with an 18-gauge cannula. Patients assessed the pain experienced using a 100-mm visual analogue score and four-point rank score. In addition, a blinded observer assessed the patient's response to venous cannulation using a four-point rank score. Good analgesia was obtained in all groups and there was no statistically significant difference in pain scores between the groups.

Effects of undegradable protein and supplemental fat on milk yield and composition and physiological responses of cows.

Because some previous studies indicated that addition of dietary fat may delay milk yield response and that effects carry over after withdrawal, an objective of this reversal design with four 28-d periods was to estimate residual effects. Diets were fed 2 wk before period 1 to permit inclusion of pretreatment diet in the mathematical model and changed for each of 33 cows at the start of periods 1 through 3; period 4 treatments continued those for period 3. Diets were 50% corn silage supplemented to be 12% CP with soybean meal and urea; 15% CP with soybean meal, blood and soybean meals, or feather and soybean meals; and 18% CP with soybean meal or blood and soybean meals. Protein treatments were replicated in diets containing 2.0% Ca soaps of fatty acids. No carry-over effects were significant; however, yield increases from Ca soaps were not evident until wk 4. Diet CP had a positive linear effect on milk and SCM yields, BW, and blood urea N. Milk protein percentage was higher from soybean meal diets. Addition of dietary Ca soaps of fatty acids increased milk, protein, fat, and SCM yields. Milk protein percentage was depressed when Ca soaps of fatty acids were fed with blood meal but not with soybean meal. No positive responses were observed from increasing dietary undegradable protein with blood meal or feather meal.

Temporal and spatial immunolocalization of cytokines in murine chronic granulomatous tissue. Implications for their role in tissue development and repair processes.

BACKGROUND: Cytokines have profound effects on various aspects of granulomatous tissue formation. However, there is little information regarding their distribution during tissue development. This study investigated the temporal and spatial distribution of transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1) and IL-1 beta in developing granulomatous tissue. EXPERIMENTAL DESIGN: Murine chronic granulomatous air pouches were induced and full thickness biopsies taken at intervals up to 28 days. Samples were prepared for immunohistochemistry and labeled using antibodies against TGF-beta, bFGF, PDGF, EGF, TNF-alpha, IL-1 alpha and IL-1 beta. RESULTS: Immunoreactivity to TGF-beta, PDGF, TNF-alpha, IL-1 alpha and IL-1 beta was localized to a proportion of macrophages within the granulomatous tissue. Immunopositive macrophage numbers increased with time, and with the exception of PDGF were associated with areas of fibrogenesis between days 14 to 28. Heterogeneous labeling of capillaries for EGF was observed within the granulomatous tissue juxtaposed to dermal musculature. Diffuse labeling of bFGF, associated with extracellular matrix, was always observed. After day 14, bFGF immunoreactivity was discretely localized to endothelial cells and the basement membrane of vessels within the granulomatous tissue. TGF-beta immunoreactivity was also associated with extracellular matrix components, being most intense in the area of fibrogenesis between 14 and 28 days. Occasional fibroblasts were also labeled with TGF-beta in this region. CONCLUSIONS: The spatial and temporal confinement of the individual cytokines suggests that a sequential coordinated process of repair and fibrosis is occurring. It is hoped that these observations will provide a more effective therapeutic approach for the sequential application of cytokines in abnormalities of wound healing.

Effect of endothelin-1 on croton oil-induced granulation tissue in the rat. A pharmacologic and immunohistochemical study.

BACKGROUND: To date no attempts have been made to determine the role of the endothelial cell derived product, endothelin-1 (ET-1) in granulation tissue development. This study investigates the cellular immunolocalization of ET-1 and its pharmacologic effect on myofibroblast-mediated rat croton oil-induced granulation tissue contraction. EXPERIMENTAL DESIGN: The distribution, cellular localization and temporal production of ET-1 in the tissues was determined by immunohistochemistry at days 7, 14, 21, and 28. The contractile response of the granulation tissue to ET-1 was tested over the same time period, and it effects modified by use of calcium antagonists. The pharmacologic profile was correlated to the ultrastructural development of contractile fibroblast-like cells within the tissue. RESULTS: Endothelin-1 caused reversible concentration-dependent contraction of the granulation tissue. The 21-day granulation tissue was the most responsive, with a maximum increase in tension of 458.9 +/- 41.1 mg; this response could be inhibited by use of calcium antagonists. Of the calcium antagonists tested, verapamil (1 x 10(-4) M) was the most potent inhibitor, giving a 43% reduction in maximum amplitude of the response. It is suggested that entry of extracellular calcium via the L-type potential operated calcium channel, is involved in ET-1 induced responses in contractile fibroblast-like cells or myofibroblasts. Ultrastructural analysis showed a correlation between the pharmacologic sensitivity of the tissue and the development of contractile fibroblast-like cells. The number of cells expressing the phenotypic characteristics of a myofibroblast increased with time, and were first observed at day 7. Immunohistochemistry revealed the presence of increasing numbers of ET-1 labeled cells throughout the time course of study. The ET-1 positive cells were localized to the capillaries. Immunolabeling of serial sections with the rodent endothelial cell specific lectin, Bandeiraea simplicifolia isolectin B4 and factor VIII-related antigen, confirmed the specific localization of ET-1 to endothelial cells. CONCLUSIONS: We present evidence that ET-1 may be an endogenous modulator of myofibroblast-mediated granulation tissue contraction and that the use of calcium antagonists could afford a possible therapeutic control in the treatment of fibrocontractive diseases.

Effects of long-term laxative treatment on neuropeptides in rat mesenteric vessels and caecum.

In order to investigate the toxic effects of long-term treatment with anthraquinone laxatives, rats were fed either chocolate alone, or chocolate adulterated with senna or danthron (1,8-dihydroxyanthraquinone) for 5 months. Mesenteric blood vessels and the outer muscle layers of the caecum, together with the myenteric plexus, were examined using ultrastructural, histochemical, immunohistochemical and immunoassay techniques. There was no ultrastructural evidence of degeneration in either the mesenteric vessels or the caecum. In the mesenteric vessels, levels of neuropeptide Y were significantly reduced in the danthron-fed rats, but levels of substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were unaffected by all treatments. In the caecum, VIP-, SP- and CGRP-immunoreactivity and catecholamine-fluorescence were unchanged by the laxative treatments.

Preliminary study to assay plasma amethocaine concentrations after topical application of a new local anaesthetic cream containing amethocaine.

Plasma concentrations of amethocaine were measured after topical application of amethocaine cream 2 g (5% w/w) to the dorsum of the right hand of 10 adult volunteers. The cream was applied for 240 min and plasma was assayed for amethocaine and its metabolite p-n-butylaminobenzoic acid at 0, 30, 60, 90, 120 and 240 min in all 10 volunteers, and at 360 min in seven volunteers, by high pressure liquid chromatography. No amethocaine was detected in the plasma of seven volunteers. Plasma concentrations of amethocaine up to 0.20 mg litre-1 were observed in three volunteers. No significant side effects were seen and pain scores on insertion of a 16-gauge cannula were 0 in all subjects. We conclude that the absence of clinical toxicity in the 10 healthy volunteers was a reflection of slow absorption and tissue hydrolysis of amethocaine after topical dermal application.

Optic disc topography in glaucoma.

An optic disc with glaucomatous cupping is said to be characterized by its extensive excavation, pallor and displacement of disc vessels. All of these features, however, can occur in normal discs. The factors which influence these features in normal eyes are reviewed and an attempt is made to define the differential diagnostic features of the disc in glaucoma.