Direct Radioimmunoassay Research Articles

Effect of dietary supplementation of Vitamin E on the resumption of cyclic ovarian activity in Murrah buffaloes

The postpartum anestrus and silent estrus are the challenging cause for low reproductive efficiency in buffaloes especially in tropical climate conditions. This study was aimed at assessing the effect of dietary supplementation of vitamin E on the resumption of cyclic ovarian activity in periparturient Murrah buffaloes through plasma progesterone (P4) hormone profiles. The Murrah buffaloes (n=12) were selected during their late gestation based on the expected date of calving that fall between November and February, from ICAR-National Dairy Research Institute livestock herd and were divided randomly into two groups (n=6). Buffaloes of group I were given only the control diet, while group II was supplemented with 2000 IU/day/head vitamin E along with control feed. Blood samples were collected from each buffalo at weekly intervals from day +21 to +56 after calving. Plasma P4 was estimated by direct radioimmunoassay (RIA). The results revealed that plasma P4 levels were statistically non-significant between groups except on day 21. However, the overall P4 profile of each buffalo indicated ovarian activity; from day 28 postpartum in supplemented group and day 42 in the control group. From the experiment, it could be inferred that the dietary supplementation of vitamin E has no effect on the resumption of cyclic ovarian activity/ steroidogenesis.

Peroxisome proliferator-activated receptor expression in the canine endometrium with cystic endometrial hyperplasia-pyometra complex.

The most common uterine diseases in bitches occurring during diestrus are cystic endometrial hyperplasia (CEH) and pyometra. These diseases can coexist as CEH-pyometra complex (CEH-P). Their pathogenesis has not been fully explained. Peroxisome proliferator-activated receptors (PPARs) are important factors regulating mammalian reproductive function and inflammatory processes. Although there is a lack of data concerning the expression of PPARs in the canine endometrium during CEH and CEH-P, we hypothesized that they might be involved in the development of pathological disorders of the canine endometrium. Therefore, the current study was conducted to evaluate and compare PPARα, PPARδ and PPARγ mRNA expression using quantitative real-time PCR (qPCR) and their immunolocalization using immunohistochemistry (IHC) staining in the endometrium of clinically healthy bitches (control group; n=8) and those with CEH (n=8) or CEH-P (n=8). For quantification, the arithmetic means of all intensities of immunostaining from the cells were measured with the optical density. PPARα, PPARδ and PPARγ were detected in the luminal epithelium, glandular epithelium and stromal cells. The mRNA transcription of PPARα was higher in the CEH group than in the control group (p<.05). Additionally, the mRNA expression and immunostaining intensities of PPARδ and PPARγ in the endometrium in the CEH-P group were downregulated relative to those in the control group (p<.05). Moreover, the serum progesterone concentration measured by direct radioimmunoassay was decreased in the CEH-P group compared to the control group (p<.001) and CEH group (p<.05). The obtained results indicate that PPARs are present in the canine endometrium and that their mRNA profile and intensity levels change under pathological conditions such as CEH and CEH-P. This finding may suggest a correlation between changes in the PPAR expression profile and hormonal disturbances, as well as the potential involvement of PPARs in signal transduction during inflammatory processes occurring in the endometrium during CEH-P. These results pave the way to further research into the role of PPARs in the pathogenesis of CEH and CEH-P in female dogs.

Diurnal Rhythm of Plasma Melatonin Concentration in the Domestic Turkey and Its Regulation by Light and Endogenous Oscillators.

Simple SummaryEnvironmental light regulates a wide range of phenomena in almost all organisms on Earth. Daily and seasonal changes in the photoperiod duration are the most important factors controlling the secretion of melatonin (MLT), a pineal hormone that affects many physiological processes in birds. The results of previous studies on the effect of MLT on the productivity and health of poultry have been promising. However, there are very few studies on the daily profiles of plasma MLT concentrations in domestic birds; therefore, we decided to examine plasma MLT levels in 10-week-old domestic turkeys exposed to different light conditions. The results demonstrated that plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark cycle showed a prominent diurnal rhythm. Night-time light exposure caused a rapid decrease in plasma MLT concentrations. The housing of turkeys in continuous dim red light revealed endogenously generated diurnal rhythm of MLT secretion. The rhythm of the plasma MLT level in a reversed cycle of 12 h dark: 12 h light adapted quickly to the new lighting condition. The aim of this study was to characterize the diurnal rhythm of plasma melatonin (MLT) concentration and its regulation by light and endogenous oscillators in 10-week-old domestic turkeys. Three experiments were performed to examine (i) the course of daily changes in plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark (12L:12D) cycle; (ii) the influence of night-time light exposure lasting 0.5, 1, 2, or 3 h on the plasma MLT level; and (iii) the occurrence of circadian fluctuations in plasma MLT levels in birds kept under continuous dim red light and the ability of turkeys to adapt their pineal secretory activity to a reversed light-dark cycle (12D:12L). The plasma MLT concentration was measured with a direct radioimmunoassay. The plasma MLT concentration in turkeys kept under a 12L:12D cycle changed significantly in a daily rhythm. It was low during the photophase and increased stepwise after the onset of darkness to achieve the maximal level in the middle of the scotophase. Next, it decreased during the second half of the night. The difference between the lowest level of MLT and the highest level was approximately 18-fold. The exposure of turkeys to light during the scotophase caused a rapid, large decrease in plasma MLT concentration. The plasma MLT concentration decreased approximately 3- and 10-fold after 0.5 and 1 h of light exposure, respectively, and reached the day-time level after 2 h of exposure. In turkeys kept under continuous darkness, the plasma MLT level was approximately 2.5-fold higher at 02:00 h than at 14:00 h. In birds kept under 12D:12L, the plasma MLT level was significantly higher at 14:00 h than at 02:00 h. The results showed that plasma MLT concentrations in 10-week-old turkeys have a prominent diurnal rhythm, which is endogenously generated and strongly influenced by environmental light.

Roles of Direct Photoreception and the Internal Circadian Oscillator in the Regulation of Melatonin Secretion in the Pineal Organ of the Domestic Turkey: A Novel In Vitro Clock and Calendar Model.

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3–6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.

Pineal gland calcification under hypoxic conditions

The pineal gland (glandula pinealis) is neuroendocrine gland located at the epithalamus of the brain secreting melatonin. The aim of this study was to explore effects of prenatal hypoxia in rats at the age of 33 weeks on the occurrence of pineal gland calcification. Distribution and chemical composition of calcerous material by light, scanning and transmission electron microscopy was investigated. Melatonin concentrations in blood plasma by direct radioimmunoassay were measured. Rats were exposed to prenatal hypoxia for 12 h at day 20 of development and second group to prenatal hypoxia for 2x8 h at days 19 and 20 of development. Vacuoles of intracellular edema in the pineal samples after 12 h hypoxia were found. Their size ranges up to 30 µm. Some of them were filled with the flocculent and fibrous material. Samples of pineal glands after 2 x 8 h hypoxia revealed the pericellular edema of pinealocytes. The amount of calcium rich particles in 2 x 8 h hypoxia group was lower than in 12 h hypoxia group. Plasma melatonin levels did not differ between control and both hypoxia groups. We concluded that calcification is a process induced by osteoblasts and osteocytes with melatonin as a promotor and it is favored under hypoxic conditions.

Association between DNA methylation of the KITLG gene and cortisol levels under stress: a replication study

A recent study reported for the first time, that DNA methylation of the KITLG gene mediates the association between childhood trauma and cortisol stress reactivity. Our study aimed to provide the first independent replication of these findings. ESPRIT is a prospective study of community-dwelling participants (age ≥ 65), randomly selected from the electoral rolls of the Montpellier district, in France. Clinical depression was assessed using the Mini-International Neuropsychiatric Interview (MINI, French version 5.00), and the Centre for Epidemiological Studies Depression Scale (CES-D). Experiences of childhood adversity were ascertained via a 25-item questionnaire. Morning, evening, and diurnal salivary cortisol was measured under basal and stress conditions and determined using direct radioimmunoassay analysis. DNA methylation of the KITLG gene was quantified in whole blood using the SEQUENOM MassARRAY EpiTYPER platform. A significant negative association was observed between KITLG DNA methylation and both morning cortisol (β = −1.846 ± 0.666, p = .007) and diurnal cortisol (area under curve [AUC]) (β = −19.429 ± 8.868, p = .031) under a stress condition. However, only the former association was significant after correcting for multiple testing. Further, this association remained after adjusting for age, sex, and depression status. No significant association was observed between childhood trauma and KITLG DNA methylation in this older population. This study provides support for an association between KITLG methylation and stress cortisol levels, suggesting that DNA methylation of this gene may play a role in the longer term regulation of the stress system.Lay summary The significant negative association between KITLG DNA methylation and morning cortisol, measured under a stressful condition, suggests that individuals with higher KITLG methylation will secrete lower levels of cortisol whilst under stress.

Preparation and development of solid phase radioimmunoassay system for determination of cortisol

ABSTRACTThe goal of this article was oriented to prepare stable polystyrene coated tubes for direct radioimmunoassay (RIA) of human cortisol. Coating process was performed using rabbit polyclonal antisera specific for cortisol. The stability study showed that these tubes could be stored for up to one year at 4ºC without any appreciable reduction in their binding. Labeling of cortisol was carried out using chloramin-T method and the 125I-cortisol tracer was purified using HPLC column. Preparation of polyclonal antibodies (anti-cortisol) was carried out in host rabbit animals against cortisol-21-hemisuccinate:Bovine serum albumin conjugate. The statistical analysis revealed good correlations between the results from the present study and the commercial IZOTOP RIA kit. The local may be extremely helpful in diagnosis and proper management of adrenal cortex disorders.

Polycystic Ovary Syndrome: An Apparently Simple yet Challenging Diagnosis.

Polycystic Ovary Syndrome: An Apparently Simple yet Challenging Diagnosis.

IgE To Penicillins With Different Specificities Can Be Identified By a Multiepitope Macromolecule. Bihaptenic Penicillin Structures and IgE Specificities

Quantitation of specific IgE by immunoassay is a recommended in vitro test for the diagnosis of immediate hypersensitivity reactions to betalactams (BLs), particularly when skin test results are negative.IgEantibodiesthatrecognizethe commonnuclear structure ofall BLsor the specificside chain structure can be mainly distinguished by immunoassays. The aim of this study was to develop an immunoassay system to detect IgE antibodies with different specificities. Cellulose discs conjugated with benzylpenicillin (BP), amoxicillin (AX) or both drugs, with poly-L-lysine (PLL) as carrier molecule, were used as solid phases in the radioallergosorbent test (RAST). Direct and inhibition radioimmunoassay studies were made to verify the structures recognized by serum IgE antibodies from penicillin-allergic patients. Our results indicated that the addition of both haptens did not decrease the capacity to capture IgE when serum specific to either BP or AX was used, at least in terms of sensitivity. In addition, the inclusion of two haptens improved significantly the levels of IgE detection in patients who recognized both BP and AX. Therefore, the use of a solid phase with a carrier molecule conjugated with two determinants (AX and BP) is helpful torecognize IgE antibodiesagainst eitherofthese determinantsandisuseful for screening sera with different specificities.

Peculiar observations in measuring testosterone in women treated with oral contraceptives supplemented with dehydroepiandrosterone (DHEA)

Total testosterone is considered to be decreased during the use of combined oral contraceptives. There is, however, considerable concern about the quality of testosterone assays, especially at low levels. We aimed to confirm testosterone levels measured by direct radioimmunoassay in a recent clinical trial with a state-of-the-art LC–MSMS method.Surplus specimens with known testosterone levels collected during the study (Clinical Trial Registration number ISRCTN06414473) were reanalyzed with an LC–MSMS method. This method was compared to another LC–MSMS method that had shown to concur excellently to a reference method. Follow-up experiments were designed to explain the results.In contrast to our expectation, LC–MSMS measurements did not corroborate the data obtained by radioimmunoassay. Subsequent experiments showed that this could be attributed to a strong dependency of the radioimmunoassay on SHBG. Testosterone results (n=198) obtained by direct radioimmunoassay showed a negative correlation to SHBG levels (r=−0.676; p<0.001). By contrast, testosterone results obtained by LC–MSMS were not related to SHBG (r=0.100; NS).In conclusion, our results indicate that total testosterone measurements during oral contraceptive use are unreliable when performed with assays sensitive to the SHBG concentration. The discrepancy with the literature can most likely be explained by the sensitivity of the immunoassay used to SHBG. Given the sharp increase in SHBG during the use of many oral contraceptives, total testosterone may not decrease, whereas its bioavailability, estimated by free testosterone levels, will be diminished. Studies aiming at restoration of testosterone homeostasis during oral contraception need to take this into account.

Assessment of theca cell function: a prerequisite to androgen or luteinizing hormone supplementation in poor responders

Poor responders are a heterogeneous population, with some patients displaying a diminished ovarian reserve and others a poor ovarian reserve with preserved granulosa cell function. Androgen and LH/hCG supplementation has been advocated for poor responders, mainly those >40 years old. Although androgens synergistically act with FSH to support folliculogenesis, and ovarian androgen secretion declines with age, there is still no evidence that androgen therapy is actually effective to improve ovarian FSH sensitivity. The main reason seems to be that theca cell function has not been appropriately assessed in patients at risk of poor response. The definition of theca insufficiency is hampered by methodologic shortcomings in routine bioassays. Provocative tests for theca cells might help to identify those patients who could benefit from androgen supplementation. The lack of data regarding theca cells in these patients might contribute to explaining the absence of evidence for a positive effect of androgen therapy.

Human phase response curve to a 1 h pulse of bright white light

The phase resetting response of the human circadian pacemaker to light depends on the timing of exposure and is described by a phase response curve (PRC). The current study aimed to construct a PRC for a 1 h exposure to bright white light (∼8000 lux) and to compare this PRC to a <3 lux dim background light PRC. These data were also compared to a previously completed 6.7 h bright white light PRC and a <15 lux dim background light PRC constructed under similar conditions. Participants were randomized for exposure to 1 h of either bright white light (n=18) or <3 lux dim background light (n=18) scheduled at 1 of 18 circadian phases. Participants completed constant routine (CR) procedures in dim light (<3 lux) before and after the light exposure to assess circadian phase. Phase shifts were calculated as the difference in timing of dim light melatonin onset (DLMO) during pre- and post-stimulus CRs. Exposure to 1 h of bright white light induced a Type 1 PRC with a fitted peak-to-trough amplitude of 2.20 h. No discernible PRC was observed in the <3 lux dim background light PRC. The fitted peak-to-trough amplitude of the 1 h bright light PRC was ∼40% of that for the 6.7 h PRC despite representing only 15% of the light exposure duration, consistent with previous studies showing a non-linear duration–response function for the effects of light on circadian resetting.

CSF levels of hypocretin-1 (orexin-A) peak during early infancy in humans.

Hypocretin (orexin) is a unique neuropeptide involved in the consolidation of wakefulness and sleep. Although hypocretin-1 levels in the cerebrospinal fluid (CSF) are stable after infancy, how levels change in preterm and term human infants is unknown. Hypocretin-1 levels were measured in CSF samples, obtained from 284 preterm (25-37 gestational weeks) and full-term infants in the first 4 months of life and 35 older children (ages 0.5-13 years), in a tertiary hospital. Detailed clinical and laboratory data were collected for each of the 319 participants. Based on that data, 108 neurologically intact children were selected (95 infants [43 preterm and 52 term] and 13 older children). CSF hypocretin-1 was measured by direct radioimmunoassay. Hypocretin-1 levels at the first weeks of the 3rd embryonic trimester (gestational age [GA] 28-34 weeks) were 314 ± 65 pg/mL (n = 17). The levels linearly increased during the third trimester and early infancy (r = 0.6), peaking in infants of 2-4 months ages (476 ± 72 pg/mL; n = 16) and decreasing thereafter; hypocretin levels in 2- to 4-month-old infants were significantly higher than those in children 0.5-13 years old (353 ± 78 pg/mL, n = 13; P = 0.0001). The present findings indicate that in human infants, CSF hypocretin-1 increases during the third embryonic trimester and is highest at 4 months of life. Thereafter, and consistent with previously published results, hypocretin levels are lower and stable until the geriatric age. This pattern may reflect the role of hypocretin in the dramatic process of sleep and wakefulness consolidation that occurs during early infancy.

Correlation between sperm characteristics and testosterone in bovine seminal plasma by direct radioimmunoassay

The objectives of this study were to validate a non-extractive RIA for seminal testosterone and quantify the hormone using a solid-phase commercial kit, and study the correlation between testosterone in seminal plasma and sperm characteristics. Parallelism showed a correlation index r = 0.992 (Y = -5.47 + 1.073X; R² = 0.985), indicating that the non-extractive method presented is indicated particularly for assessment of testosterone when establishing comparisons between samples. Overall mean (±SD) of testosterone level was 0.60±0.65 ng/mL. Correlation was only found between the seminal concentrations of testosterone and pH of the semen.

Abstract A3: Premenopausal serum androgens and breast cancer risk: A nested case-control study

Abstract Introduction: Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited. Methods: A case-control study nested within a prospective cohort, the NYU Women's Health Study, was conducted. A total of 356 cases (276 invasive and 80 in situ) and 683 individually-matched controls (1:2 matching) were included. Matching variables included age and date of blood donation, as well as phase and day of menstrual cycle. Testosterone, androstenedione, dehydroandrosterone sulfate (DHEAS), and sex hormone-binding globulin (SHBG) were measured using direct radioimmunoassays. Free testosterone was calculated. Samples from a second blood donation at a median of 2 years after the first blood donation were also analyzed for 138 cases and 268 controls. Results: Premenopausal serum testosterone and free testosterone concentrations were positively associated with subsequent breast cancer risk. In models adjusted for known risk factors of breast cancer (BMI, age at menarche, family history of breast cancer, parity/age at first birth, and history of breast biopsy), the odds ratios for increasing quintiles of testosterone were 1.0 (reference), 1.5 (95% confidence interval (CI), 0.9–2.3), 1.2 (95% CI, 0.7–1.9), 1.4 (95% CI, 0.9–2.3) and 1.8 (95% CI, 1.1–2.9) with the P value for trend of 0.04, and for free testosterone were 1.0 (reference), 1.2 (95% CI, 0.7–1.8), 1.5 (95% CI, 0.9–2.3), 1.5 (95% CI, 0.9–2.3), and 1.8 (95% CI, 1.1–2.8) with a P value for trend of 0.01). A marginally significant positive association was observed with androstenedione (p = 0.07), but no association with DHEAS or SHBG. Results were consistent in analyses stratified by tumor type (invasive, in situ), estrogen receptor status, age at blood donation, and menopausal status at diagnosis. Intra-class correlation coefficients (ICCs) for measurements of serial annual blood donations from the same individual were greater than 0.7 for all biomarkers except for androstenedione (0.57 in controls). Conclusions: Premenopausal concentrations of testosterone and free testosterone are positively associated with breast cancer risk. Results from other prospective studies are consistent with our results. Testosterone and free testosterone are also highly reliable (i.e. a single measurement is reflective of a woman's average level over time). Future studies should examine the contribution of including testosterone and free testosterone in breast cancer risk prediction models for women between the ages of 40 and 50. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A3.

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

High prevalence of hypovitaminosis D in pregnant Japanese women with threatened premature delivery

Serum 25-hydroxyvitamin D (25-OHD) concentrations are thought to accurately reflect vitamin D stores, and vitamin D deficiency causes secondary hyperparathyroidism, irreversible bone loss, and increased risk of fracture. Recent studies suggest that decrease of serum 25-OHD level in mothers could increase the risk of preeclampsia, cesarean section, and craniotabes. Furthermore, this deficiency may affect bone mass and the incidence of neuromuscular diseases of their children in the future. In the present study, the serum concentration of 25-OHD in 93 pregnant women after the 30th week of their gestation was determined by direct radioimmunoassay. Mean 25-OHD levels in spring, summer, fall, and winter were 14.3 ± 5.1, 15.7 ± 6.4, 13.7 ± 5.1, and 13.9 ± 4.2 ng/ml, respectively. Severe vitamin D deficiency (25-OHD < 10 ng/ml) was found in 10 of these 93 women. Overall, hypovitaminosis D, which was defined as serum 25-OHD concentration equal to or less than 20 ng/ml, was revealed in 85 mothers (89.5%). Serum 25-OHD levels were not associated with either intact parathyroid hormone or corrected calcium concentrations, but were negatively associated with serum type I collagen N-terminal telopeptide and bone-specific alkaline phosphatase in these subjects. Mothers with threatened premature delivery had significantly lower 25-OHD levels (11.2 ± 3.2 ng/ml) than those in mothers with normal delivery (15.6 ± 5.1 ng/ml). In conclusion, the present data suggest a high prevalence of hypovitaminosis D in perinatal pregnant Japanese women throughout the year, which seems to affect bone metabolism and to be associated with threatened premature delivery.

血漿および気管支-肺胞洗蘇液 (BALF) 中腺性カリクレインの直接ラジオイムノアッセイ法の開発とその臨床応用

血漿および気管支-肺胞洗蘇液 (BALF) 中腺性カリクレインの直接ラジオイムノアッセイ法の開発とその臨床応用

Estriol and the Kidney in Pregnancy

Estriol-16α-glucosiduronate in the urine and serum of pregnant women was measured by a direct radioimmunoassay which was highly specific for this compound and did not require hydrolysis or chromatography. Urinary and serum estriol-16α-glucosiduronate increased as pregnancy progressed. Pregnant women with presumptive renal failure (toxemia of pregnancy, malignant hypertension, post renal transplantation, etc) showed low urinary levels of estriol-16α-glucosiduronate but normal serum levels. Their renal clearance of estriol-16α-glucosiduronate was calculated provisionally and compared to that of creatinine. The results suggest that some serum estriol is conjugated by the kidney and that the measurement of estriol-16α-glucosiduronate can be used as a clinical index of renal failure during pregnancy.

Development and validation of a high throughput direct radioimmunoassay for the quantitative determination of serum and plasma melatonin (N-acetyl-5-methoxytryptamine) in mice

A simple, sensitive and specific high throughput radioimmunoassay (RIA) for the quantitative determination of total melatonin (N-acetyl-5-methoxytryptamine) was developed. This method allows the analysis of melatonin in different biological fluids with small sample volumes (e.g. mice and rats), and wide working range. With the preparation of matrix-specific calibrators called "Equalizing Reagent" the influence of results due to different composition between standards and sample matrix was reduced. This reagent is produced by use of the respective biological liquid. Endogenous melatonin is removed by adsorption to activated charcoal. The melatonin-free biological liquid is then used to equalize the assay matrix of standards and untreated samples. Finally, all samples including the standards are digested by use of a protease to reduce non-specific binding, for example to albumin or albumin-like molecules. High-affinity specific antibodies were produced by immunization of rabbits with 5-methoxytryptamine-bovine serum albumin. The review of cross reactions to ten structurally similar compounds showed that the antibody has a high specificity for melatonin. This direct RIA uses a [(125)I]-melatonin tracer for the determination of melatonin. 5-methoxytryptamine was synthesized by direct iodination with [(125)I]-Bolton-Hunter-Reagent. The required acceptance criteria for validation parameters were fulfilled. The flexible standards cover a working range from 12 to 4000 pg/mL with a sample volume of 50 microL (e.g. working range from 3 to 1000 pg/mL with a sample volume of 200 microL). The limit of detection in mouse serum and mouse plasma was 9 pg/mL and 7 pg/mL, respectively. The recovery of melatonin in mouse serum was 108% and in mouse plasma 99%. The variation coefficients of the assay, within and between runs, ranged between 7 and 13% in mouse serum and between 5 and 8% in mouse plasma. Based on the determination of a 24-h profile of melatonin in mouse samples a characteristic diurnal rhythm of melatonin was observed. The wide working range makes it possible to analyse low and high melatonin concentrations. The validated direct RIA was compared with established sample preparation methods such as liquid-liquid- and solid-phase-extraction followed by RIA. The correlation for methanol extraction is y=1.1x-0.9, R(2)=0.98, P<0.001 and C(18)-extraction y=0.8x-0.03, R(2)=0.99, P<0.001. The distinct advantage of the direct assay of melatonin is that complicated extraction steps can be avoided. The realized advantages of the direct RIA when compared to a commercially available melatonin RIA are its low sample volume and ease of implementation. Exemplary, plasma melatonin was determined at C3H, C57BL, wild-type and melatonin receptor (MT1-/- and MT2-/-) knockout mice kept under L:D=12:12 cycles. The results have indicated that at all mouse strains have plasma melatonin content, with higher levels during the night and lower levels during the day. Because of different melatonin concentrations the direct RIA convince by its low detection limit and wide working range. The determination of serum and plasma melatonin in mice by ELISA or HPLC-technique previously failed due to the required use of a high sample volume and the low sensitivity. In summary, it can be concluded that the developed and validated direct RIA meets the requirements for the determination of melatonin and is especially suitable for the analysis of melatonin in different mouse strains.