Determination of cannabinoids in human cerumen samples by use of UPLC-MS/MS as a potential biomarker for drug use.

A quantitative analytical procedure was developed and validated by the use of Ultra- Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) for the determination of Cannabidiol (CBD), Cannabinol (CBN), Δ9-Tetrahydrocannabinol (Δ9-THC), Cannabichromene (CBC), Cannabigerol (CBG) and 11-Nor- 9- Carboxy- Tetrahydrocannabinol (THC-COOH) in an unconventional biological matrix, cerumen. All the investigated calibration curves were characterized by high correlation values (R2 ≥ 0.9965). The LODs and LOQs ranged from 0.004 to 0.009 µg g-1 and 0.012-0.029 µg g-1, respectively. Intra-assay and inter-assay precision were found to be 0.6-2.5%, and 0.8-2.2%, respectively. All recovery values of cannabinoids, with the use of the optimum cotton swab, at low (0.008 µg g-1 of cerumen), medium (0.037 µg g-1of cerumen) and high (0.16 µg g-1 of cerumen) control levels, were estimated to be above 86%. The method developed here permitted the analysis of real cerumen samples obtained from fourteen cannabis users. In twelve out of fourteen cases, Δ9-THC was found to be positive, while in six cases, three major cannabinoids, CBN, CBG and Δ9-THC were quantified at concentrations 0.02-0.21 µg g-1, 0.01-0.24 µg g-1 and 0.01-4.86 µg g-1, respectively. Subject #8 has the highest amount of the detected substances in both left and right ear, with Δ9-THC at a concentration of 1.85 and 4.86 µg g-1, CBG 0.06 and 0.24 µg g-1, CBN 0.10 and 0.21 µg g-1, respectively. In addition, a detection window for the substances Δ9-Tetrahydrocannabinol, Cannabinol and Cannabigerol, in cerumen, was defined with success. In this case, Δ9-THC reached a maximum detection frame of up to fifteen days after smoking 0.5 g of marijuana cigarette. ANOVA-one-way analysis also indicated that the average earwax production of non-cannabis users differs significantly from the one of cannabis users (p = 0.048, <0.05). On the other hand, no significant difference was noticed between male and female users as the p value exceeded 0.05. In addition, no significant effect was observed on earwax production in regard to age, frequency and the last time of use (p > 0.05). These last three factors proved to have a significant impact on cannabinoids concentrations, since p values were less than 0.05.

The (TTTA)n polymorphism of aromatase (CYP19) gene is associated with age at menarche.

BACKGROUND: Twin studies have shown that age at menarche may be subject to hereditary influences but the specific determinants are unknown. Estrogens are known to have an important role in menarche. Since the enzyme aromatase is responsible for the conversion of androgens to estrogens, the aromatase (CYP19) gene could be a candidate gene for the regulation of menarche. The aim of this study was to investigate the possible association of the CYP19(TTTA)(n) polymorphism with age at menarche. METHODS: We studied 130 healthy adolescent females from a closed community in North-Western Greece. Information on menarche was obtained through interviews. The BMI was recorded. The CYP19(TTTA)(n) polymorphism was genotyped. RESULTS: The mean age at menarche was 12.9 ± 1.2 years and the BMI = 19.8 ± 2.3 kg/m(2). Genotype analysis revealed 5 CYP19(TTTA)(n) alleles containing 7-11 TTTA repeats. Girls homozygous for the allele with 7 TTTA repeats had earlier menarche (12.45 ± 0.9 years) than girls carrying other genotypes (13.0 ± 1.2 years, P = 0.025), whereas the BMI was not different between these two subgroups. Carriers of the allele with 11 TTTA repeats had later menarche compared with non-carriers (14.1 ± 0.75 versus 12.8 ± 1.2 years, P< 0.001), whereas no difference was found in BMI values. Comparing girls with early menarche (<12 years, 25th percentile) with girls with late menarche (>13.75 years, 75th percentile), we found that 31% of the girls with early menarche were homozygous for the (TTTA)(7) allele compared with 6.9% among girls with late menarche (P = 0.018). In addition, none of the girls carrying the (TTTA)(11) allele was found among the subgroup with early menarche, whereas 24.1% of girls with late menarche had the (TTTA)(11) allele (P = 0.001). No association between other alleles and age at menarche was found. CONCLUSIONS: There is evidence for a genetic contribution of the CYP19 gene to the age at menarche.

Cerebral cavernous malformations: congruency of histopathological features with the current clinical definition.

AIM: Cerebral cavernous malformations (CCMs) are defined as a mulberry-like assembly of thin walled vascular sinusoids lined by a thin endothelium lacking smooth muscle and elastin, displaying no intervening brain parenchyma. In this study, we analyse the congruency of histopathological features with the current clinical definition on a large series of neuroradiologically verified CCMs. METHODS: 87 patients who received no primary treatment prior to surgery were included. Preoperative MRIs of all patients were reviewed. 12 histopathological parameters were assessed systematically, using haematoxylin-eosin, Prussian blue, elastica van Gieson and congo red for amyloid detection. RESULTS: 71/87 (81.6%) of the cases fulfilled the basic histological criteria of CCMs. However, the thickness of the vessel walls and the calibre of the malformed vessels were highly variable. 16/87 cases (18.4%) were histologically non-diagnostic. Non-diagnostic specimens were significantly associated with radiological signs of haemorrhage (p = 0.001). A few cases (4.6%) regionally contained capillary-like malformed vessels. Intervening brain parenchyma between malformed vessels throughout the lesion was seen in 50/71 (70.4%) diagnosable lesions. Haemosiderin deposits, gliosis, thrombosis, fibrotic changes, hyalinised vessel walls, calcification and cholesterol crystals were present in a considerable range. In addition, we found amyloid deposits in 14/87 (16.1%) specimens. CONCLUSION: Contrary to the current clinical definition, the absence of intervening brain parenchyma does not represent an essential histopathological criterion of CCMs in our series. Furthermore, the diameter of the vessel lumina and the thickness of vessel walls varied considerably. Based on these findings, adaptation of the current definition on the basis of interdisciplinary interaction needs to be considered.

High-field, high-resolution, susceptibility-weighted magnetic resonance imaging: improved image quality by addition of contrast agent and higher field strength in patients with brain tumors.

INTRODUCTION: To demonstrate intratumoral susceptibility effects in malignant brain tumors and to assess visualization of susceptibility effects before and after administration of the paramagnetic contrast agent MultiHance (gadobenate dimeglumine; Bracco Imaging), an agent known to have high relaxivity, with respect to susceptibility effects, image quality, and reduction of scan time. METHODS: Included in the study were 19 patients with malignant brain tumors who underwent high-resolution, susceptibility-weighted (SW) MR imaging at 3 T before and after administration of contrast agent. In all patients, Multihance was administered intravenously as a bolus (0.1 mmol/kg body weight). MR images were individually evaluated by two radiologists with previous experience in the evaluation of pre- and postcontrast 3-T SW MR images with respect to susceptibility effects, image quality, and reduction of scan time. RESULTS: In the 19 patients 21 tumors were diagnosed, of which 18 demonstrated intralesional susceptibility effects both in pre- and postcontrast SW images, and 19 demonstrated contrast enhancement in both SW images and T1-weighted spin-echo MR images. Conspicuity of susceptibility effects and image quality were improved in postcontrast images compared with precontrast images and the scan time was also reduced due to decreased TE values from 9 min (precontrast) to 7 min (postcontrast). CONCLUSION: The intravenous administration of MultiHance, an agent with high relaxivity, allowed a reduction of scan time from 9 min to 7 min while preserving excellent susceptibility effects and image quality in SW images obtained at 3 T. Contrast enhancement and intralesional susceptibility effects can be assessed in one sequence.

High-resolution contrast-enhanced, susceptibility-weighted MR imaging at 3T in patients with brain tumors: correlation with positron-emission tomography and histopathologic findings.

BACKGROUND AND PURPOSE: The purpose of this work was to demonstrate susceptibility effects (SusE) in various types of brain tumors with 3T high-resolution (HR)-contrast-enhanced (CE)-susceptibility-weighted (SW)-MR imaging and to correlate SusE with positron-emission tomography (PET) and histopathology. MATERIALS AND METHODS: Eighteen patients with brain tumors, scheduled for biopsy or tumor extirpation, underwent high-field (3T) MR imaging. In all of the patients, an axial T1-spin-echo (SE) sequence and an HR-SW imaging sequence before and after IV application of a standard dose of contrast agent (MultiHance) was obtained. Seven patients preoperatively underwent PET. The frequency and formation of intralesional SusE in all of the images were evaluated and correlated with tumor grade as determined by PET and histopathology. Direct correlation of SusE and histopathologic specimens was performed in 6 patients. Contrast enhancement of the lesions was assessed in both sequences. RESULTS: High-grade lesions demonstrated either high or medium frequency of SusE in 90% of the patients. Low-grade lesions demonstrated low frequency of SusE or no SusE. Correlation between intralesional frequency of SusE and histopathologic, as well as PET, tumor grading was statistically significant. Contrast enhancement was equally visible in both SW and SE sequences. Side-to-side comparison of tumor areas with high frequency of SusE and histopathology revealed that intralesional SusE reflected conglomerates of increased tumor microvascularity. CONCLUSIONS: 3T HR-CE-SW-MR imaging shows both intratumoral SusE not visible with standard MR imaging and contrast enhancement visible with standard MR imaging. Because frequency of intratumoral SusE correlates with tumor grade as determined by PET and histopathology, this novel technique is a promising tool for noninvasive differentiation of low-grade from high-grade brain tumors and for determination of an optimal area of biopsy for accurate tumor grading.

Combined estrogen receptor alpha and estrogen receptor beta genotypes influence the age of menarche.

BACKGROUND: Age at menarche has a strong genetic influence. We reported recently an association between the XbaI (351A-->C)and PvuII (397T-->C) polymorphisms of the estrogen receptor (ER)alpha gene with the age of menarche in Greek adolescents. In the present study, we examined whether ERbeta genotypes alone, or in combination with ERalpha genotypes, may also influence onset of menarche. METHODS: We performed genotyping for the single nucleotide polymorphisms 1730A-->G and 1082G-->A of the ERbeta gene and examined their association with the age of menarche in the same cohort of 145 Greek girls. We then looked for a possible effect of combined ERalpha and beta genotypes on the age of menarche. RESULTS: Menarche occurred 7 months later in girls with the AA genotype of the 1730A-->G polymorphism than in girls with the AG genotype (mean +/- SD: 13.23 +/- 1.24 versus 12.66 +/- 1.26 years, respectively; P = 0.005). The 1082G-->A polymorphism was not detected in any of the girls examined. A significant effect of combined ERalpha and beta genotypes was also apparent. Menarche occurred 11 months later in girls bearing the AA/TT,AA (ERalpha, ERbeta) genotypes compared with girls with the CC/CC,AG genotype (13.30 +/- 1.27 nersus 12.41 +/- 1.28 years; P = 0.042). The difference remained significant after adjusting for body mass index (P = 0.034). CONCLUSION: Combined ERalpha and ERbeta polymorphisms may influence the age of menarche.

Association of SHBG gene polymorphism with menarche.

The age of menarche may be subject to hereditary influences but the specific determinants are unknown. Our aim was to investigate the possible association of a functional (TAAAA)n polymorphism in the promoter of the sex hormone-binding globulin (SHBG) gene with the timing of menarche. This polymorphism has been associated with polycystic ovary syndrome (PCOS) and is considered to contribute to SHBG levels. We studied 130 healthy normal-weight adolescent females from a closed community in North-Western Greece. Information on menarche was obtained through interviews. The BMI was recorded. Genomic DNA was isolated from peripheral blood leukocytes for genotyping the TAAAA repeat region. We subdivided our subjects into two groups based on median age of menarche: those with menarche <13 years and those with menarche > or =13 years. Genotype analysis revealed six (TAAAA)n alleles containing 5-10 TAAAA repeats. The distribution of alleles was different in the two groups. Girls with late menarche had more frequently longer TAAAA alleles (>8 repeats), while girls with early menarche had shorter alleles at a greater frequency (P=0.048). The major contribution to early menarche was by the 6 TAAAA repeat allele. Furthermore, carriers of the longer allele genotypes had later menarche (13.24+/-1.15 years) than those with shorter allele genotypes (12.67+/-1.15, P=0.018). These findings provide evidence for a genetic contribution of SHBG gene to the age of menarche.

Association of polymorphisms of the oestrogen receptor alpha gene with the age of menarche.

BACKGROUND: The age of menarche may be subject to hereditary influences, but the specific genetic determinants are largely unknown. We evaluated whether the XbaI and PvuII polymorphisms of the estrogen receptor alpha gene are associated with the age of menarche. METHODS: We performed genotyping for XbaI and PvuII in a cohort of 145 adolescent females from a closed community in North-Western Greece. RESULTS: There was strong linkage disequilibrium between the two polymorphisms. Menarche occurred later in girls with the XX genotype than in girls with the Xx or xx genotype (mean +/- SD: 13.36 +/- 1.24 versus 12.80 +/- 1.14 and 12.75 +/- 1.35 years respectively; P = 0.017). Menarche also tended to occur later in PP homozygotes than in Pp and pp subjects, but the difference was not significant (mean +/- SD: 13.09 +/- 1.29 versus 12.80 +/- 1.19 and 12.85 +/- 1.33 years respectively). The strongest effect was seen when the PX haplotype was considered [mean +/- SD: 13.43 +/- 1.18 years for homozygotes versus 12.76 +/- 1.25 years in heterozygotes and in subjects without the PX allele, P = 0.006]. CONCLUSIONS: We document that the XbaI polymorphism, and possibly PvuII, may be genetic determinants of the age of menarche.