An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow's Milk).

Ochratoxins are mycotoxins that have been extensively studied lately due to the multiple toxic effects such as nephrotoxicity, hepatotoxicity, and carcinogenicity. These toxins contaminate plant and animal foods and after ingestion they reach into body fluids. The method of competitive direct enzyme immunoassay, in the solid phase, was validated through the determination of specific parameters (performance, linearity, recovery percentage, limit of detection, limit of quantification). The validated method was used to determine ochratoxin A in colostrum and cow's milk. The method applied for the determination of ochratoxin A was linear for the concentration range of 0.0-0.5 ng/mL, the value for the regression coefficient (r) was 0.9838. Ochratoxin A was present in 91.67% of the colostrum and in 93.33% of cow's milk samples. The linearity of the method, demonstrated for very low concentrations of analyte, the detection limit as well as the limit of quantification recommend the method for the determinations of micro-pollutants from foods, including biological fluids.

Magnetic particles-based enzyme immunoassay for rapid determination of secoiridoid glycoside, amarogentin.

Amarogentin (AG) is one of the bitter secoiridoid glycosides, which exerts various pharmacological activities as a bitter stomachic. Recently, there is an increasing demand for AG-containing plants in Japan due to their use as folk medicines and food additives; hence, it is crucial to develop analytical techniques that are specific for AG. In this study, a new magnetic particles-based enzyme immunoassay (MPs-EIA) using a specific monoclonal antibody against AG (MAb 1E9) for the rapid determination of AG in plants of the family Gentianaceae was described. AG directly immobilized onto magnetic particles (MPs) was used as a competitor for free AG against MAb 1E9, thereby increasing the surface area of the solid phase and decreasing the immunoreaction time. In addition, the blocking step required in case of the conventional enzyme-linked immunosorbent assay could be avoided in the proposed MPs-EIA, which enables an even more rapid performance for the immunoassay. In the developed MPs-EIA, AG exhibited linearity in the range of 15.6-500 ng mL-1, with a limit of detection of 8.58 ng mL-1. Validation analysis revealed that MPs-EIA is a sufficiently sensitive and rapid for the quantitative analysis of AG in plant samples. To the best of our knowledge, this is the first MPs-EIA that has been applied to plant samples.

The evaluation of an immunoperoxidase assay applicable in antiviral drug screening.

Bovine viral diarrhea virus (BVDV) fall into cytopathic (CP) and noncytopathic (NCP) biotypes, based on their ability to kill cultured cells. NCP-BVDV can not be titrated by conventional means as used for CP-BVDV, which has impeded the identification of antiviral drugs targeting NCP-BVDV virus strains. In this study, the application of an immunoperoxidase assay in the screening of antiviral drugs was tested using two known BVDV inhibitors, ribavirin and ammonium chloride (NH4Cl). Phospholipase C inhibitor U73122 was identified to affect BVDV infection by using this immunoperoxidase assay. In addition, the results of immunoperoxidase assay were validated by real-time PCR. Taken together, the immunoperoxidase assay is a useful and versatile method suitable for antiviral drug screening targeting NCP-BVDV.

Intra-individual variability in TSH levels of healthy women during the first half of pregnancy / Variabilidad intra-individual de los niveles TSH en mujeres sanas en primer trimestre de gestación

Objective: TSH is the parameter most widely accepted to assess thyroid function, especially in pregnant women. The aim of this current study was to analyze intra-individual changes in TSH during the first half of pregnancy in women with TSH levels higher than 2.5mIU/L in early pregnancy. Methods: An observational, prospective study was conducted on 243 healthy pregnant women in the first trimester of pregnancy. Thyroid function was assessed by testing TSH and free T4 levels. A subgroup of women with TSH levels >2.5mIU/L underwent additional tests (TSH, free T4, peroxidase antibodies). Information on dietary iodine intake and/or iodine supplements was also recorded. Results: Mean TSH level was 1.89mIU/L (range 0.024-6.48mIU/L), and mean FT4 level was 1.19ng/dL (range 0.80-1.90ng/dL). Fifty-eight women (23.8%) had TSH levels>2.5mIU/L in the first trimester of pregnancy, and additional thyroid function tests were performed in 27 women. TSH levels significantly decreased from the first to the second test (3.59±0.92mIU/L vs 2.81±1.06mIU/L respectively; p<0.01), and the decrease was significantly greater in pregnant women who used iodized salt as compared to those who did not (1.16±0.65mIU/L vs 0.19±0.93mIUI/L respectively; p<0.01). A positive correlation was found between the time elapsed to the second measurement (24.3±17.2 days; range 8-58) and the decrease in TSH levels (r=0.40; p=0.038). Conclusion: TSH levels showed a continuous, uniform decrease during the first half of pregnancy in women with values slightly above the normal range. Pregnant women who used iodized salt were more likely to have decreased TSH levels in a second test (AU)

Objetivo: La TSH es el parámetro más aceptado para evaluar la función tiroidea, especialmente en mujeres embarazadas. El objetivo del presente estudio fue analizar los cambios intraindividuales de TSH durante la primera mitad de la gestación, en aquellos casos en los que la TSH en las primeras etapas de la gestación fue superior a 2,5 mUI/L. Métodos: Estudio observacional prospectivo que incluyó a 243 mujeres embarazadas sanas en el primer trimestre de gestación. Se estudió función tiroidea mediante TSH y T4 libre. Un subgrupo de mujeres con TSH> 2,5 mUI/L fueron sometidas a un segundo análisis (TSH, T4 libre, anticuerpos antiperoxidasa). También se registró información sobre la ingesta de yodo con la dieta y/o suplementos. Resultados: La TSH media fue de 1,89 mUI/L (rango 0,024-6,48 mUI/L), y la T4 libre media fue de 1,19 ng/dL (rango 0,80-1,90ng/dL). El 23,8% (58 mujeres) presentaron TSH> 2,5 mUI/L en el primer trimestre de gestación, realizándose una segunda valoración en 27 pacientes. La TSH disminuyó significativamente del primer al segundo análisis (3,59±0,92 mUI/L vs. 2,81±1,06 mUI/L respectivamente, p <0,01). La TSH disminuyó significativamente más en aquellas mujeres embarazadas que consumieron sal yodada que en aquellas que no lo hicieron (1,16±0,65 mUI/L vs. 0,19±0,93 mUI/L respectivamente, p<0,01). Hubo una correlación positiva entre el tiempo transcurrido para una segunda determinación (24,3±17,2 días, rango 8-58 días), y la reducción en los niveles de TSH (r= 0,40; p=0,038). Conclusión: La disminución de los niveles de TSH con la edad gestacional es uniformemente continua a lo largo de la primera mitad de la gestación en aquellos casos con TSH ligeramente por encima del rango sugerido de normalidad. Las mujeres embarazadas que consumían sal yodada tenían más probabilidades de reducir los niveles de TSH en un segundo análisis (AU)

Electroacupuncture inhibits excessive interferon-γ evoked up-regulation of P2X4 receptor in spinal microglia in a CCI rat model for neuropathic pain.

BACKGROUND: Although electroacupuncture (EA) is effective in the relief of neuropathic pain, the underlying mechanisms remain unclear. Previous studies have reported immunomodulatory effects of EA in rats. Since excessive release of interferon-γ (IFN-γ) after nerve injury transforms quiescent spinal microglia into an activated state with more neuropathic pain, associated with purinergic receptor P2X4 expression, it is possible that EA may mediate its analgesic effect by attenuating IFN-γ release and subsequent generation of P2X4R(+) microglia. METHODS: Male rats underwent chronic constriction injury (CCI) or IFN-γ intrathecal injection and von Frey tests were performed to evaluate the effect of EA on pain thresholds. Spinal IFN-γ and P2X4R expression levels were measured by immunohistochemistry, real-time PCR, enzyme immunoassay, and/or western blots. In vitro primary cultures of microglia were used to examine IFN-γ activation of P2X4R(+) cells. RESULTS: In CCI rats, EA treatment significantly increased paw withdrawal threshold relative to control. IFN-γ facilitated P2X4R(+) microglia activation both in vitro and in vivo. EA also down-regulated both P2X4R and IFN-γ expression in the spinal cord after CCI. However, EA did not exert the same analgesic effect after intrathecal IFN-γ injection. CONCLUSIONS: EA ameliorated tactile allodynia after peripheral nerve injury by down-regulating excessive expression of IFN-γ in the spinal cord and subsequently reducing expression of P2X4R.

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N-acetyl-seryl-aspartyl-lysyl-proline.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N-terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP-like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using ß-D-galactosidase-labeled Gly-γAbu-SDKP as a marker antigen, an anti-rabbit IgG-coated immunoplate as a bound/free separator and 4-methylumbelliferyl-ß-D-galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP-IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter-assay and 13.3 and 7.8% for intra-assay comparisons. Plasma AcSDKP-IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients.

Cyclooxygenase expression and prostaglandin levels in central nervous system tissues during the course of chronic relapsing experimental autoimmune encephalomyelitis (EAE).

OBJECTIVE: Multiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and orchestrates both diseases. One particular group of mediators are the prostaglandins (PGs), which we have previously shown, through quantitation and pharmacological intervention, to be closely involved in the pathology of MS and EAE. The aim of the current study was to determine the expression of the PG-generating cyclooxygenase (COX) enzymes and the profile of PGE(2) and PGD(2), in selected central nervous system (CNS) tissues, with the development of the chronic relapsing (CR) form of EAE. In particular, the work investigates the possible relationship between the expression of COX isoenzymes and PG levels during the neurological phases of CR EAE. METHODS: CR EAE was induced in Biozzi mice with inoculum containing lyophilised, syngeneic spinal cord emulsified in complete Freund's adjuvant. The cerebral cortex, cerebellum and spinal cord were dissected from mice during the acute, remission and relapse stages of disease with a minimum of five animals per treatment. The expression of COX-1, COX-1b variant and COX-2, in pooled samples, was determined by Western blotting. PGE(2) and PGD(2) levels in extracted samples were measured using commercial enzyme immunoassay kits. RESULTS: COX-2 expression in spinal cords during acute disease remained unaltered and was in contrast to an enhancement of the enzyme, together with COX-1 and COX-1b, in all other sampled areas. PGE(2) and PGD(2) levels remained unchanged during the acute phase and the subsequent remission of symptoms. COX-1 and COX-1b expression was elevated in tissues during the relapse stage of CR EAE and concentrations of the prostanoids were markedly increased. CONCLUSIONS: The study examines the implications of COX isoenzyme expression over the course of CR EAE and discusses the reported relationship between PGE(2) and PGD(2) in the instigation and resolution of CNS inflammation. Consideration is also given to the treatment of CR EAE and suggests that drugs designed to limit the inflammatory effects of the PGs should be administered prior to or during the relapse phase of the disease.

Effects of cocoa extract and dark chocolate on angiotensin-converting enzyme and nitric oxide in human endothelial cells and healthy volunteers--a nutrigenomics perspective.

Evidence suggests that cocoa from the bean of Theobroma cacao L. has beneficial effects on cardiovascular disease. The aim of this study was to investigate if cocoa extract and dark chocolate influence angiotensin-converting enzyme (ACE) and nitric oxide (NO) in human endothelial cells (in vitro) and in healthy volunteers (in vivo). ACE activity was analyzed with a commercial radioenzymatic assay and measured in human endothelial cells from umbilical veins (HUVEC) after 10 minutes of incubation with cocoa extract. NO was measured after 24 hours of incubation. ACE activity and NO were measured at baseline and after 30, 60, and 180 minutes in 16 healthy volunteers after a single intake of 75 g of dark chocolate containing 72% cocoa. Significant inhibition of ACE activity (P < 0.01) and significant increase of NO (P < 0.001) were seen in HUVEC. In the study subjects, a significant inhibition of ACE activity (mean 18%) 3 hours after intake of dark chocolate was seen, but no significant change in NO was seen. According to ACE genotype, significant inhibition of ACE activity was seen after 3 hours in individuals with genotype insertion/insertion and deletion/deletion (mean 21% and 28%, respectively). Data suggest that intake of dark chocolate containing high amount of cocoa inhibits ACE activity in vitro and in vivo.

Determination of cAMP in plant cells by a modified enzyme immunoassay method.

Presently, there is no doubt about the functioning of the adenylate cyclase signaling system in plants, but the role of this system in various physiological-biochemical processes has been investigated insufficiently. Cyclic adenosine monophosphate (cAMP), the key component produced by adenylate cyclase, whose concentrations in plant cells vary rather widely, is the indicator of functional activity for this signaling way. In the latter case, in the process of determination of concentrations of this messenger, one encounters difficulties related to insufficient sensitivity of the methods most frequently applied. In this connection, the proposed mechanism is a modification of the method of the enzyme immunoassay (EIA), which is based on immediate measurement of cAMP concentrations in the sample with the use of antibodies. This modification allows us to determine the concentrations of cAMP with the precision of 5 pM, which exceeds the sensitivity of other methods by approximately 10 times. The specificity of the assay has been confirmed by other two independent tests--the capillary electrophoresis and the nuclear magnetic resonance (NMR). It has also been compared to the data obtained with the use of the commercial kit from Sigma-Aldrich. The modification has been tested on such plant objects as in vitro potato plants, and suspension cells of potato and Arabidopsis.

Ginsenosides chemistry, biosynthesis, analysis, and potential health effects.

Ginsenosides are a special group of triterpenoid saponins that can be classified into two groups by the skeleton of their aglycones, namely dammarane- and oleanane-type. Ginsenosides are found nearly exclusively in Panax species (ginseng) and up to now more than 150 naturally occurring ginsenosides have been isolated from roots, leaves/stems, fruits, and/or flower heads of ginseng. Ginsenosides have been the target of a lot of research as they are believed to be the main active principles behind the claims of ginsengs efficacy. The potential health effects of ginsenosides that are discussed in this chapter include anticarcinogenic, immunomodulatory, anti-inflammatory, antiallergic, antiatherosclerotic, antihypertensive, and antidiabetic effects as well as antistress activity and effects on the central nervous system. Ginsensoides can be metabolized in the stomach (acid hydrolysis) and in the gastrointestinal tract (bacterial hydrolysis) or transformed to other ginsenosides by drying and steaming of ginseng to more bioavailable and bioactive ginsenosides. The metabolization and transformation of intact ginsenosides, which seems to play an important role for their potential health effects, are discussed. Qualitative and quantitative analytical techniques for the analysis of ginsenosides are important in relation to quality control of ginseng products and plant material and for the determination of the effects of processing of plant material as well as for the determination of the metabolism and bioavailability of ginsenosides. Analytical techniques for the analysis of ginsenosides that are described in this chapter are thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) combined with various detectors, gas chromatography (GC), colorimetry, enzyme immunoassays (EIA), capillary electrophoresis (CE), nuclear magnetic resonance (NMR) spectroscopy, and spectrophotometric methods.

Avaliação da imunidade passiva em bezerros nascidos de vacas imunizadas com vacina contra rotavírus / Evaluation of passive immunity in calves born from cows immunized with anti-rotavirus vaccine

Com o objetivo de monitorar a imunidade passiva em bezerros alimentados com colostro de vacas imunizadas e não imunizadas com vacina contra rotavírus, foram determinados títulos de anticorpos em amostras de sangue e colostro de 26 vacas da raça Holandesa no dia do parto e de seus bezerros, à zero, às 24, 48 horas e aos sete, 14, 21, 28 dias de idade, pelo ensaio imunoenzimático. Tanto no soro sangüíneo como no colostro, os títulos dos isótipos IgG, IgG1 e IgG2 foram mais elevados no grupo dos animais vacinados, porém somente no colostro o aumento foi significativo. Os bezerros alimentados com o colostro das vacas vacinadas apresentaram títulos mais altos dos isótipos IgG, IgG1, IgG2, IgA e IgM, após a ingestão do colostro, sendo constatado aumento significativo apenas para os títulos do isótipo IgG2. Amostras positivas para rotavírus foram detectadas nos dois grupos experimentais a partir dos sete dias de idade. A vacinação materna não protegeu efetivamente os bezerros das infecções naturais por rotavírus, pois, apesar de aumentar os títulos séricos de anticorpos anti-rotavírus nos animais vacinados, não foi capaz de impedir a ocorrência da rotavirose nos bezerros alimentados com o colostro das vacas imunizadas.

Passive immune response in calves fed colostrum from immunized and nonimmunized cows by anti-rotavirus vaccine was monitored. Titers of antibodies were determined by immunoenzymatic assay in blood and colostrum sampled at parturition day from 26 Holstein cows as well as in blood from their calves collected at 0, 24, and 48 hours and seven, 14, 21, and 28 days after birth. In serum and colostrum, IgG, IgG1, and IgG2 antibody titers were higher in vaccinated animals; however, this increase was only significant in colostrum. The calves fed colostrum from vaccinated cows showed higher IgG, IgG1, IgG2, IgA, and IgM isotypes titers after the ingestion of colostrum, being evidenced significant increase only for IgG2 titers. Positive samples for rotavirus were detected in both experimental groups since seven days after birth. Results showed that maternal vaccine failed to protect effectively the calves from natural infections by rotavirus, though it increased the anti-rotavirus antibody titers in vaccinated animals, but was not capable to impair the occurrence of rotaviruses in the calves fed colostrum from immunized cows.

Panax ginseng induces anterograde transport of pigment organelles in Xenopus melanophores.

Melanophores from Xenopus laevis are pigmented cells, capable of quick colour changes through cyclic adenosine 3':5'-monophosphate (cAMP) coordinated transport of their intracellular pigment granules, melanosomes. In this study we use the melanophore cell line to evaluate the effects of Panax ginseng extract G115 on organelle transport. Absorbance readings of melanophore-coated microplates, Correlate-EIA direct cAMP enzyme immunoassay kit, and western blot were used to measure the melanosome movement and changes in intracellular signalling. We show that Panax ginseng induces a fast concentration-dependent anterograde transport of the melanosomes. No significant increase in the cAMP level was seen and pre-incubation of melanophores with the protein kinase C (PKC) inhibitor EGF-R Fragment 651-658 (M-EGF) only partly decreased the ginseng-induced dispersion. We also demonstrate that Panax ginseng, endothelin-3 (ET-3) and alpha-melanocyte stimulating hormone (MSH) stimulate an activation of mitogen activated protein kinase (MAPK). Pre-incubation with M-EGF decreased the MAPK activity induced by ET-3 and MSH, but again only marginally affected the response of Panax ginseng. Thus, in melanophores we suggest that Panax ginseng stimulates an anterograde transport of pigment organelles via a non-cAMP and mainly PKC-independent pathway.

[Adaptation of different modifications of enzyme immunoassay and immune blot assay to assess the candidate vaccine HIVREPOL against HIV/AIDS].

A preclinical trial of the vaccine HIVREPOL provided a complex of methods for assessing the identity and specific activity of vaccines against HIVIAIDS. The identity of "HIVREPOL" has been assessed by indirect enzyme immunoassay (EIA): the vaccine specifically binds the antibodies of the sera from HIV-infected individuals. Immune blot assay was the most informative method for assessing the identity of the candidate vaccine. The sera from HIVREPOL-vaccinated mice recognized the proteins gp41, p24, p55 of cultured HIV1 on "New-Lay-Blot1" strips. The bands corresponding to p24 were revealed in the line blots "Blot-HIV-1/2+O" and "INNO-LIA-HIV-Confirmation". The specific activity of the HIVREPOL vaccine was confirmed from the reactivity of sera of the mice vaccinated with recombinant proteins of the immunosorbents available in EIA test systems for the detection of HIV antibodies. Competitive EIA established the antigen-binding activity of sera from HIVREPOL-vaccinated mice against the native reference HIV-1 antigen.

Prostaglandin-H-synthase (PGHS)-1 and -2 microtiter assays for the testing of herbal drugs and in vitro inhibition of PGHS-isoenzyms by polyunsaturated fatty acids from Platycodi radix.

In order to test inhibition of prostaglandin-H-synthase-1 and -2 (PGHS-1 and -2) by plant extracts, we have established two enzyme based in vitro assays with enzyme immunoassay (EIA) evaluation. The assays have been evaluated with known synthetic inhibitors and with plant extracts. In a screening of traditionally used Chinese herbs for anti-inflammatory activity, a series of n-hexane and dichloromethane extracts showed significant inhibitory effect in comparison with the known specific PGHS-2 inhibitors NS-398 (IC(50) = 2.6 microM) and nimesulide (IC(50) = 36 microM). The lipophilic extracts of the Chinese drug Jiengeng, the dried roots of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae), showed good inhibitory activity against both PGHS isoenzymes. The directly prepared DCM-extract exhibited better activity against PGHS-2 (IC(50) = 4.0 microg/ml) than against PGHS-1 (IC(50) = 17.6 microg/ml). We identified fatty acids as main active constituents and quantified them. Linoleic acid showed the highest content (ca. 20% of the dried extract) and a high and preferential PGHS-2 inhibitory activity (IC(50) (PGHS-1) = 20 microM; IC(50) (PGHS-2) = 2 microM). The comparison of the concentration of linoleic acid and the inhibitory activity of the direct DCM-extract showed, that linoleic acid is mainly responsible for the in vitro activity of the extract on PGHS-2.

Value of an enzymatic assay for the determination of serum ceruloplasmin.

The serum concentration of the copper protein ceruloplasmin has been an important diagnostic indicator of Wilson's disease (WD). It is widely quoted that 95% of people with WD have low serum ceruloplasmin concentrations. Current evidence suggests that a normal serum ceruloplasmin concentration is more common in patients with WD, particularly those with liver disease, perhaps in part because of the routine use of an immunologic assay. This assay might indicate a normal level of ceruloplasmin when the enzymatic activity is lower. Enzymatic activity is the biologically relevant parameter. We compared the immunologic measurement with the enzymatic assessment of oxidase activity in patients with liver or neurologic symptoms of unknown origin in whom WD was considered in the differential diagnosis. Although a strong correlation of ceruloplasmin protein concentration with oxidase activity was observed in controls, this was not the case for these patients. Twelve patients, presenting with various types of hepatic disease, demonstrated a weak correlation between ceruloplasmin protein concentration and oxidase activity. Ten percent of patients with neurologic symptoms ( n = 41) had low ceruloplasmin concentrations and oxidase activity, and another 8% had normal ceruloplasmin concentrations associated with low oxidase activity. Although the enzymatic method is preferred for its biologic relevance, ceruloplasmin analysis is not a reliable diagnostic parameter for the diagnosis of WD in patients with liver disease. An important use of the ceruloplasmin oxidase assay is in the follow-up of patients with WD. Ceruloplasmin oxidase activity was undetectable in sera from patients with WD who were undergoing long-term chelation therapy, suggesting an early sign of copper depletion and a need for subsequent monitoring for symptoms of copper deficiency.

Analysis methods of ginsenosides.

Ginsenosides are considered the main active principles of the famous Chinese traditional medicine "ginseng". For more than 30 years many researchers developed methods for the identification and quantification of ginsenosides in ginseng plant material, extracts and products. Separation of ginsenosides has been achieved using thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). Among these techniques HPLC is by far the most employed. Ultraviolet (UV), evaporative light scattering (ELSD), fluorescence and, recently, mass spectrometry (MS) were coupled with HPLC for the detection of ginsenosides. The most recent methods are here discussed together with a critical evaluation of the published results. Furthermore new techniques such as near infrared spectroscopy (NIRS) and enzyme immunosassay (EIA) recently used for the determination of ginsenosides will be discussed.

Recent advances in enzyme assays.

Enzyme assays for high-throughput screening and enzyme engineering, which are often based on derivatives of coumarin, nitrophenol, fluorescein, nitrobenzofurazane or rhodamine dyes, can be divided into two categories: those that depend on labelled substrates, and those that depend on sensing the reactions of unmodified substrates. Labelled substrates include, for example, fluorogenic and chromogenic substrates that generate a reporter molecule by beta-elimination, fluorescence resonance energy transfer (FRET) substrates and isotopic labels for enantioselectivity screening. By contrast, endpoint sensing can be done using amine reagents, fluorescent affinity labels for phosphorylated proteins, or synthetic multifunctional pores. Sensing assays can also be done in real time by using, for example, aldehyde trapping to follow vinyl ester acylation in organic solvent or calcein-copper fluorescence for sensing amino acids. The current trend is to assemble many such assays in parallel for enzyme profiling and enzyme fingerprinting.

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.

Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants.

Gonadotropin-releasing hormone (GnRH) is critical for the initiation and maintenance of reproduction in vertebrates. Information regarding GnRH release is abundant in mammals, but absent in poikilothermic tetrapods. In this study, we established a novel GnRH enzyme immunoassay (EIA) to measure GnRH release over time from hypothalamic explants isolated from mature field-caught and commercially-acquired male bullfrogs, Rana catesbeiana. Hypothalamic explants from rats were used as a positive control to test the sensitivity and accuracy of our EIA and to ensure our in vitro system could detect GnRH pulses. Prominent GnRH pulses were present in the majority (9/10) of rat hypothalamic explants, but absent in all (17/17) of the commercial bullfrogs and the majority (5/8) of field-caught bullfrogs. In three cases where GnRH pulses were observed in field-caught bullfrogs, there was only one pulse during the 2-h incubation period; high-frequency pulses similar to those observed in rats were not observed. Veratridine, which opens voltage-gated sodium channels, stimulated GnRH release in all explants cultured in the presence of Ca(2+), demonstrating explant viability. The levels of both spontaneous and veratridine-induced GnRH release were significantly higher in field-caught than commercial bullfrogs. This study demonstrated, for the first time, the temporal pattern of GnRH release in a poikilothermic tetrapod. Further, our results suggest the levels and patterns of GnRH output in bullfrogs are subject to the dynamic regulation by physiological and environmental cues.

Enzyme immunoassays for the investigation of protein nitration by air pollutants.

Two enzyme immunoassays have been developed, characterised, and applied to investigate protein nitration in birch pollen extract (BPE) and bovine serum albumin (BSA) samples exposed to air pollutants. The monoclonal antibody CAY-189542 against nitrotyrosine (raised against peroxynitrite-treated keyhole limpet hemocyanine) was characterised in an indirect competitive assay (affinity and cross-reactivities) and applied in a new one-sided enzyme immunoassay for nitrated proteins. The one-sided assay was calibrated against a nitrated BSA standard with an average of 14 nitrotyrosine residues per molecule (nitro-(14)-BSA; detection limit 8.3 pmol L(-1)), and the sensitivity of the test was found to be significantly enhanced by a multivalent binding mode of the monoclonal antibody (bonus effect of multivalency). The same antibody and a polyclonal antibody against Bet v 1, the most prominent birch pollen allergen, were used in a new sandwich immunoassay for specific determination of nitrated Bet v 1. This assay was calibrated against a nitrated Bet v 1 standard with an average of 3 nitrotyrosine residues per molecule (nitro-(3)-Bet v 1; detection limit 0.2 nmol L(-1)). Bet v 1 and BSA exposed to polluted urban outdoor air and to synthetic gas mixtures containing NO2 and O3 at atmospherically relevant concentration levels were found to be efficiently nitrated within hours to days. Pronounced correlations of nitro-(14)-BSA equivalent concentrations with exposure time and with nitro-(3)-Bet v 1 equivalent concentrations in nitrated BPE samples were observed. Test experiments indicated that the efficiency of protein nitration was strongly enhanced by reactive species formed upon interaction of NO2 with O3 and H2O (e.g. NO3 and HNO3). Potential implications of protein nitration by air pollutants are outlined and discussed.