Tecoma stans Floral Extract-Based Biosynthesis for Enhanced Surface Plasmon-Coupled Emission and a Preliminary Study on Fluoroimmunoassay.

We demonstrate the synthesis of biogenic supported silver spiked star architectures and their application to increase the electromagnetic field intensity at its tips that enhance plasmon-coupled emission. Tecoma stans floral extract has been used to synthesize silver nanocubes and spiked stars. We observe ∼445-fold and ∼680-fold enhancements in spacer and cavity configurations, respectively, in the SPCE platform. The hotspot intensity and Purcell factor are evaluated by carrying out finite-difference time-domain (FDTD) simulations. Time-based studies are presented to modulate the sharpness of the edges wherein an increase in the tip sharpness with the increase in reaction time up to 5 h is observed. The unique morphology of the silver architectures allowed us to utilize them in biosensing application. A SPCE-based fluoroimmunoassay was performed, achieving a 1.9 pg/mL limit of detection of TNF-α cytokine. This combination of anisotropic architectures, SPCE and immunoassay prove to be a powerful platform for the ultrasensitive detection of biomarkers in surface-bound assays.

Is eosinophilic esophagitis an equivalent of pollen allergic asthma? Analysis of biopsies and therapy guided by component resolved diagnosis

Background: Eosinophilic esophagitis (EoE) is characterized by esophageal dysfunction and, histologically, by eosinophilic inflammation. There is not a clear etiologic treatment. Biopsies analysis using plant histology methods may show callose and pollen tubes in the esophageal mucosa. Component-resolved diagnosis (CRD) with microarrays could detect possible allergens involved and indicate an elimination diet and allergen immunotherapy (AIT). Methods: One hundred and twenty-nine patients with EoE were tested for environmental and food allergens. CRD, histological and botanical analysis were performed. Clinical scores and endoscopic biopsy were performed every six months for three years. Fifty healthy patients, 50 asthmatics due to pollen, and 53 celiac disease patients were included as comparison groups. CRD-directed AIT was administered in 91 EoE patients and elimination diet in 140 patients (87 EoE and all 53 CD patients). Results: CRD detected allergen hypersensitivity in 87.6% of patients with EoE. The predominant allergens were grass group 1 (55%), lipid transfer proteins (LTP) of peach and mugwort, hazelnuts and walnuts. Callose from pollen tubes was found in 65.6% of biopsies. After CRD-guided elimination diet and/or AIT, 101 (78.3%) EoE patients showed significant clinical improvement (p < 0.017) and 97 (75.2%) were discharged (negative biopsy, no symptoms, no medication) without relapse (AU)

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Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix).

The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu3+-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 µg/mL for standard SSa sample with a detection limit of 0.006 µg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.

[HTRF-based high-throughput PGE2 release prohibition model and application in discovering traditional Chinese medicine active ingredients].

Prostaglandin (PG) E2 is an active substance in pathological and physiological mechanisms, such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence(HTRF) PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps, including the cell density optimization and IC50 values determination of a positive compound, were taken to verify the stability and sensibility of the assay. Low intra-plate, inter-plate and day-to-day variability were observed in this 384-well, high-throughput format assay. Totally 5 121 samples were selected from the company's traditional Chinese medicine(TCM) material base library and used to screen PGE2 inhibitors. In this model, the cell plating density was 2 000 cells for each well; the average IC50 value for positive compounds was (7.3±0.1) µmol; the Z' factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples, 228 components exhibited a PGE2 production prohibition rate of more than 50%, and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy, indicating the reliability and authenticity of the screening results. The automated screening system was introduced to make the model fast and efficient, with a average daily screening amount exceeding 14 000 data points and provide a new model for discovering new anti-inflammatory and analgesic drug and quickly screening effective constituents of TCM in the early stage.

Defining thresholds of specific IgE levels to grass pollen and birch pollen allergens improves clinical interpretation.

BACKGROUND: Cut-off values and predictive values are used for the clinical interpretation of specific IgE antibody results. However, cut-off levels are not well defined, and predictive values are dependent on the prevalence of disease. The objective of this study was to document clinically relevant diagnostic accuracy of specific IgE for inhalant allergens (grass pollen and birch pollen) based on test result interval-specific likelihood ratios. Likelihood ratios are independent of the prevalence and allow to provide diagnostic accuracy information for test result intervals. METHODS: In a prospective study we included consecutive adult patients presenting at an allergy clinic with complaints of rhinitis or rhinoconjunctivitis. The standard for diagnosis was a suggestive clinical history of grass or birch pollen allergy and a positive skin test. Specific IgE was determined with the ImmunoCAP Fluorescence Enzyme Immuno-Assay. RESULTS: We established specific IgE test result interval related likelihood ratios for clinical allergy to inhalant allergens (grass pollen, rPhl p 1,5, birch pollen, rBet v 1). The likelihood ratios for allergy increased with increasing specific IgE antibody levels. The likelihood ratio was <0.03 for specific IgE <0.1 kU/L, between 0.1 and 1.4 for specific IgE between 0.1 kU/L and 0.35 kU/L, between 1.4 and 4.2 for specific IgE between 0.35 kU/L and 3.5 kU/L, >6.3 for specific IgE>0.7, and very high (∞) for specific IgE >3.5 kU/L. CONCLUSION: Test result interval specific likelihood ratios provide a useful tool for the interpretation of specific IgE test results for inhalant allergens.

Synergism between exposure to mercury and use of iodine supplements on thyroid hormones in pregnant women.

OBJECTIVE: To evaluate the association between mercury exposure and thyroid-stimulating hormone (TSH), total triiodothyronine (TT3) and free thyroxine (FT4) levels during pregnancy as well as to explore if there is any synergic action between mercury and intake of iodine from different sources. METHODS: The study population was 1407 pregnant women participating in the Spanish INMA birth cohort study. Total mercury concentrations were analyzed in cord blood. Thyroid hormones (THs) were measured in serum samples collected at 13.2±1.5 weeks of gestation. The association between mercury and TH levels was evaluated with multivariate linear regression models. Effect modification caused by iodine intake from supplements and diet was also evaluated. RESULTS: The geometric means of TSH, TT3, FT4 and mercury were 1.1µU/L, 2.4nmol/L, 10.5pmol/L and 7.7µg/L, respectively. Mercury levels were marginally significantly associated with TT3 (ß: -0.05; 95%CI: -0.10, 0.01), but were neither associated with TSH nor FT4. The inverse association between mercury and TT3 levels was stronger among the iodine supplement consumers (-0.08; 95%CI: -0.15, -0.02, interaction p-value=0.07). The association with FT4 followed the same pattern, albeit not significant. CONCLUSION: Prenatal mercury exposure was inversely associated with TT3 levels among women who took iodine supplements during pregnancy. These results could be of public health concern, although further research is needed.

Inhibition of c-Kit signaling by diosmetin isolated from Chrysanthemum morifolium.

The interaction of stem cell factor (SCF) with its cognate receptor c-Kit is closely associated with the survival and maturation of melanocytes. To investigate novel depigmentation agents, we screened 2,000 plant extracts for c-Kit inhibitors to identify active small molecules by using time-resolved fluorescence enzyme assays. For the active extracts identified as inhibitors of c-Kit enzyme, we evaluated the effects of the active extracts and isolated flavonoids on c-Kit phosphorylation in MO7e/melanocytes. Anti-melanogenic activity was also examined in melanocytes and melanoderm model. The flavonoids such as diosmetin, apigenin, acacetin and luteolin isolated from Chrysanthemum morifolium were found to be active in inhibiting c-Kit both at enzyme and cellular levels. In addition, these flavonoids attenuated SCF-induced proliferation of human primary melanocytes without toxicity and suppressed ultraviolet (UV) B irradiation-mediated melanin synthesis significantly. Among the active flavonoids, diosmetin was found to inhibit SCF-induced melanogenesis in a human melanoderm model. These results strongly suggest that C. morifolium extract and diosmetin have potential to suppress SCF-/UVB-induced melanogenesis, and could be developed as anti-pigmentation agents.

High seroprevalence of human herpesvirus type 8 in patients with end-stage renal disease in Taiwan.

BACKGROUND: Human herpesvirus type 8 (HHV-8) is the aetiologic agent of Kaposi's sarcoma (KS). The incidence of KS in renal transplant patients is much higher than in healthy controls. The risk is even higher among recipients seropositive for HHV-8 before transplantation. Patients with end-stage renal disease (ESRD) are immunocompromised and are candidates for renal transplantation, but HHV-8 seroprevalence in ESRD patients has not been well documented. OBJECTIVES: This study aimed to evaluate HHV-8 seroprevalence in ESRD patients in a cohort in Taiwan. STUDY DESIGN: Blood samples collected from 149 ESRD patients and 149 age- and sex-matched healthy controls were analysed for HHV-8 antibody with immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) and for HHV-8 DNA with polymerase chain reaction. RESULTS: Seropositivity and titres for HHV-8 antibodies with IFA as well as seropositivity with ELISA were significantly greater in ESRD patients than in healthy controls (P=0.006, 0.001 and 0.003, respectively). Patients with a history of taking herbal medicine had significantly greater ELISA positivity than those without such a history (P=0.004). ELISA positives, particularly patients, had much higher IFA antibody titres than ELISA negatives (P<0.0001). Seropositivity in ESRD patients was not related to lymphopaenia, monocytosis, dialysis duration or a history of transfusion. Two diabetic ESRD patients were positive for HHV-8 DNA. CONCLUSIONS: ESRD patients had significantly greater HHV-8 seropositivity than healthy controls in Taiwan. This association seems to be related to the geographic location of the cohort and invites further studies for the early association of HHV-8 infection in ESRD patients and risk for KS.

Analysis of soluble CD14 and its use as a biomarker in neonatal foals with septicemia and horses with recurrent airway obstruction.

Soluble CD14 (sCD14) binds bacterial lipopolysaccharide (LPS) and acts as an anti-inflammatory LPS-inhibitor in vivo. In humans, sCD14 is one of the soluble biomarkers used for various inflammatory diseases and conditions, however, sCD14 assays have not yet been evaluated in horses. Here, we developed and optimized a bead-based assay for the quantification of sCD14 in horses. The assay was then used to determine native sCD14 concentrations in serum from healthy and septic foals, in the colostrum of healthy mares and in plasma from adult horses with recurrent airway obstruction (RAO) and control horses. Healthy foals and adult horses had sCD14 concentrations in serum or plasma in the high ng/ml range. The concentration of sCD14 in colostrum samples from healthy mares was in the µg/ml range. Foals with septicemia and adult horses with RAO had significantly higher sCD14 concentrations in their circulation than the respective control groups. The findings suggest that sCD14 can become a valuable biomarker for neonatal septicemia, RAO and possibly also for other inflammatory diseases in horses. Further studies and larger samples numbers are required to determine normal sCD14 concentration ranges and those that are indicative of disease progression, severity or prognosis.

Enhancing of intensity of fluorescence by DEP manipulations of polyaniline-coated Al2O3 nanoparticles for immunosensing.

A novel modification of low-cost Al2O3 nanoparticles (Al2O3 NPs) for antibody-protein immunosensing is proposed. The modified NPs are utilized to enhance the intensity of fluorescence in a dielectrophoretic (DEP) chip with a microelectrode array. The surface of the Al2O3 NPs is modified by ionic polyaniline (PANDB) rather than the conventional silane (3-aminopropyltrimethoxysilane) to conjugate the antibody on the outer shell. After the PANDB-Al2O3 NPs is functionalized to form probes, a DEP chip with a vertical non-uniform electric field that is produced by top and bottom electrodes condenses and immobilizes the nanoprobes on the surface of the electrodes by positive DEP force for immunosensing of the fluorescent protein. Additionally, each microelectrode array can be individually controlled with/without DEP manipulation using a computer program. Experimental results indicate that PANDB-based nanoprobes provide more rapid and sensitive immunosensing than those having undergone conventional silane modification. During immunosensing, fluorescence intensity can be doubled by the application of extra DEP force. The individual control of NPs on the microelectrode array has great potential for applications in multi-antibody arrays in a single chip for immunosensing.

Plasma enterolactone and incidence of endometrial cancer in a case-cohort study of Danish women.

The phyto-oestrogen enterolactone has been hypothesised to protect against hormone-dependent cancers, probably through its antioestrogenic potential. We investigated whether a higher level of plasma enterolactone was associated with a lower incidence of endometrial cancer in a case-cohort study in the 'Diet, Cancer and Health' cohort. The cohort study included 29 875 women aged 50­64 years enrolled between 1993 and 1997. Information on diet and lifestyle was provided by self-administrated questionnaires and blood was drawn from each participant. Time-resolved fluoroimmunoassay was used for biochemical determination of plasma enterolactone. A total of 173 cases and 149 randomly selected cohort members were included. We estimated incidence rate ratio (IRR) and 95% CI by a Cox proportional hazards model. A 20 nmol/l higher plasma concentration of enterolactone was associated with a non-significant lower risk of endometrial cancer (IRR 0.93, 95% CI 0.84, 1.04). When excluding women with low enterolactone concentrations (quartile 1) due to potential recent antibiotic use, the association became slightly stronger, but remained non-significant (IRR 0.90, 95% CI 0.79, 1.02). Menopausal status, hormone replacement therapy or BMI did not modify the association. In conclusion, we found some support for a possible inverse association between plasma enterolactone concentration and endometrial cancer incidence.

An extracellular lipid transfer protein is relocalized intracellularly during seed germination.

Plant lipid transfer proteins (LTPs) constitute a family of small proteins recognized as being extracellular. In agreement with this notion, several lines of evidence have shown the apoplastic localization of HaAP10, a LTP from Helianthus annuus dry seeds. However, HaAP10 was recently detected intracellularly in imbibing seeds. To clarify its distribution, immunolocalization experiments were performed during the course of germination and confirmed its intracellular localization upon early seed imbibition. Further assays using a hydrophobic dye, FM4-64, inhibitors of vesicular traffic, and immunolocalization of the pectin rhamnogalacturonan-II, allowed the conclusion that endocytosis is activated as soon as seed imbibition starts. Furthermore, this study demonstrated that HaAP10 is endocytosed throughout imbibition. Biochemical and cellular approaches indicate that the intracellular fraction of this LTP appears associated with oil bodies and some evidence also suggest its presence in glyoxysomes. So, HaAP10 is apoplastic in dry seeds and upon imbibition is rapidly internalized and relocalized to organelles involved in lipid metabolism. The results suggest that HaAP10 may be acting as a fatty acid shuttle between the oil body and the glyoxysome during seed germination. This concept is consistent with the initial proposition that LTPs participate in the intracellular transfer of lipids which was further denied based on their apparent extracellular localization. This report reveals for the first time the relocalization of a lipid transfer protein and opens new perspectives on its role.

A gold nanocage-CNT hybrid for targeted imaging and photothermal destruction of cancer cells.

This communication reports the design of a novel aptamer conjugated gold nanocage decorated SWCNTs hybrid nanomaterial for targeted imaging and selective photothermal destruction of the prostate cancer cells.

Singlet oxygen in Escherichia coli: New insights for antimicrobial photodynamic therapy.

Antimicrobial photodynamic therapy is an emerging treatment for bacterial infections that is becoming increasingly more attractive because of its effectiveness and unlikelihood of inducing bacterial resistance. However, there is limited knowledge about the localization of the photoactive drug in the bacteria and about the details of production of the main cytotoxic species, singlet oxygen. This article describes a combination of spectroscopic and time-resolved photophysical techniques that provide such information for a cationic porphyrin photosensitizer in gram-negative Escherichia coli bacteria. Our results reveal a double localization of the photosensitizer, inside (bound to the nucleic acids) and outside (bound to the cell wall) of the E. coli cells. Singlet oxygen is produced at both sites and is able to cross the cell wall.

Diagnosis of prostate cancer by detection of minichromosome maintenance 5 protein in urine sediments.

BACKGROUND: The accuracy of prostate-specific antigen (PSA) testing in prostate cancer detection is constrained by low sensitivity and specificity. Dysregulated expression of minichromosome maintenance (Mcm) 2-7 proteins is an early event in epithelial multistep carcinogenesis and thus MCM proteins represent powerful cancer diagnostic markers. In this study we investigate Mcm5 as a urinary biomarker for prostate cancer detection. METHODS: Urine was obtained from 88 men with prostate cancer and from two control groups negative for malignancy. A strictly normal cohort included 28 men with complete, normal investigations, no urinary calculi and serum PSA <2 ng ml(-1). An expanded control cohort comprised 331 men with a benign final diagnosis, regardless of PSA level. Urine was collected before and after prostate massage in the cancer patient cohort. An immunofluorometric assay was used to measure Mcm5 levels in urine sediments. RESULTS: The Mcm5 test detected prostate cancer with 82% sensitivity (confidence interval (CI)= 72-89%) and with a specificity ranging from 73 (CI=68-78%) to 93% (CI=76-99%). Prostate massage led to increased Mcm5 signals compared with pre-massage samples (median 3440 (interquartile range (IQR) 2280 to 5220) vs 2360 (IQR <1800 to 4360); P=0.009), and was associated with significantly increased diagnostic sensitivity (82 vs 60%; P=0.012). CONCLUSIONS: Urinary Mcm5 detection seems to be a simple, accurate and noninvasive method for identifying patients with prostate cancer. Large-scale prospective trials are now required to evaluate this test in diagnosis and screening.

Impact of a lignan-rich diet on adiposity and insulin sensitivity in post-menopausal women.

There has been a growing interest in lignans, a class of phyto-oestrogens, because of their potentially favourable effects on human health. The aim of the present study was to compare the metabolic profile of post-menopausal women consuming various amounts of dietary lignans. Phyto-oestrogen intake was assessed using a 3-d dietary record analysed with a Canadian food phyto-oestrogen content data table in 115 post-menopausal women (age 56.8 (SD 4.4) years and BMI 28.5 (SD 5.9) kg/m(2)). Plasma enterolactone (ENL), the major biologically active metabolite of dietary lignans, was determined by time-resolved fluoroimmunoassay. Anthropometrics, abdominal adipose tissue areas (computed tomography), body composition (hydrostatic weighing) and insulin sensitivity (hyperinsulinaemic-euglycaemic clamp) were measured in all women. Women in the high dietary lignan intake subgroup (n 29) had a significantly lower BMI and total body fat mass, as well as a better glucose disposal rate (GDR; P < 0.05), compared with women in the low lignan intake subgroup (n 28). The majority of women with the highest dietary lignan intake were also in the highest quartile of plasma ENL (59 %). Women in the highest ENL quartile had a significantly lower BMI (26.1 (SD 4.4) v. 30.4 (SD 6.9) kg/m(2), P < 0.05), total body fat mass (24.8 (SD 9.8) v. 33.3 (SD 13.3) kg, P < 0.05), 2 h postload glycaemia (5.5 (SD 0.9) v. 5.7 (sd 0.8) nmol/l, P < 0.05) and a higher GDR (8.3 (SD 2.7) v. 5.5 (SD 2.8), P < 0.01) compared with women in the lowest ENL quartile. In conclusion, women with the highest ENL concentrations had a better metabolic profile including higher insulin sensitivity and lower adiposity measures.

Variation in fasting and non-fasting serum enterolactone concentrations in women of the Malmö Diet and Cancer cohort.

OBJECTIVES: The aim of this study was to examine the variation of enterolactone from fasting and non-fasting blood of middle-aged healthy women eating a normal diet to determine the usefulness of a single sample in epidemiological studies. SUBJECTS AND METHODS: Twenty-six women born between 1940 and 1950 were recruited within the Malmö Diet and Cancer cohort. Three non-fasting and two overnight fasting samples were collected from each individual during a 5-week period. Twenty-one participated in all measurements. Enterolactone concentrations were analyzed by time-resolved fluoroimmunoassay. RESULTS: The within-subject and between-subject variations (coefficient of variations, CV) were estimated to 59 and 89% respectively for fasting samples and 71 and 67% for non-fasting samples. The intraclass correlation coefficients (ICC) were estimated to 0.66 (95% confidence interval (CI) 0.35-0.84) for fasting and 0.48 (95% CI, 0.22-0.72) for non-fasting samples. CONCLUSIONS: Although the estimated ICC for blood samples was moderate, it indicates that enterolactone levels of both fasting and non-fasting blood samples should be useful in future projects within the Malmö Diet and Cancer cohort.

[Analysis of detecting methods of digoxin blood drug level].

Digoxin plays a part in healing of congestive heart failure in clinic. Its therapeutic dose is very approximate to toxic dose and even they overlap each other sometimes. There are many influencing factors on blood drug level of digoxin. Pharmacodynamics and pharmacokinetics varies with different individuality. It is indispensable to detecting blood drug level in order to treat disease and prevent intoxication. Integrating with the detecting-methods of blood drug level of digoxin home and broad, characteristic of many methods are summarized from sensitivity, linearity range, cross-reaction and precision. These methods include radio immunoassay, enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay and HPLC-MS-MS. These methods are popular for their specialized ascendancy. The cost of radio immunoassay is low. Enzyme immunoassay has good specificity. Sensitivity and stability of chemiluminescence immunoassay is very excellent. Fluorescence polarization immunoassay is sensitive and convenient. HPLC-MS-MS has high resolution and good specificity. One of the development tendencies is to combine two or more methods in detecting the blood drug level of digoxin which contribute to these methods integrated use.

Profiles of alpha-melanocyte-stimulating hormone in the Japanese flounder as revealed by a newly developed time-resolved fluoroimmunoassay and immunohistochemistry.

Profiles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the Japanese flounder were examined by a newly developed time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. A TR-FIA for alpha-MSH was newly developed, and its levels in the pituitary gland and plasma of Japanese flounder reared in a white or black tank for 5 months were compared. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, alpha-MSH antibodies, samples, and europium-labeled Des-Ac-alpha-MSH. The sensitivity of the assay, defined as twice the standard deviation at a zero dose, was 0.98 ng/ml (49 pg/well). The intra- and interassay coefficients of variation of the assay were 8.8% (n=8) and 17.3% (n=5), respectively, at about 50% binding. Cross-reactivities of Des-Ac-alpha-MSH and Di-Ac-alpha-MSH were about 100%. Cross-reactivities of adrenocorticotropic hormone, salmon gonadotropin-releasing hormone (sGnRH), and chicken GnRH-II were less than 0.2%, and that of melanin-concentrating hormone was less than 2.0% at 50% binding. Displacement curves of serially twofold-diluted hypothalamus extract, pituitary gland extract, and plasma extract of Japanese flounder with the assay buffer were parallel to the alpha-MSH standard curve. Moreover, displacement curves of serially twofold-diluted hypothalamus and/or pituitary gland extract of masu salmon, goldfish, red seabream, Japanese eel, tiger puffer, and barfin flounder with the assay buffer were also parallel to the alpha-MSH standard. In Japanese flounder, total immunoreactive (ir)-alpha-MSH levels in the pituitary gland were lower in the black tank, whereas those in the plasma tended to be higher in the black tank, suggesting that the synthesis and release of alpha-MSH are higher in the black tank. alpha-MSH-ir cells were detected in the pars intermedia and a small part of the pars distalis of the pituitary gland. alpha-MSH-ir cell bodies were located in the basal hypothalamus and alpha-MSH-ir fibers were distributed not only in the hypothalamus but also in the telencephalon, midbrain, cerebellum, and medulla oblongata, suggesting that alpha-MSH functions as a neuromodulator in the brain.

Monoclonal antibody-based time-resolved fluorescence immunoassays for daidzein, genistein, and equol in blood and urine: application to the Isoheart intervention study.

BACKGROUND: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women. METHODS: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction. RESULTS: Comparison of monoclonal TR-FIA, commercial polyclonal antibody-based TR-FIA, and gas chromatography-mass spectrometry showed correlations (r, 0.911-0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied. CONCLUSIONS: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.