Interlaboratory comparison study of serum total testoserone measurements performed by mass spectrometry methods

Background

Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays.

Methods

Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis.

Results

The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range.

Conclusions

The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.     

Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina

Spirulina platensis (SP) is a filamentous cyanobacterium microalgae with potent dietary phyto-antioxidant, anti-inflammatory and anti-cancerous properties. The present study aimed to investigate the chemopreventive effect of SP against rat liver toxicity and carcinogenesis induced by dibutyl nitrosamine (DBN) precursors, and further characterized its underlying mechanisms of action in HepG2 cell line. Investigation by light and electron microscopy showed that DBN treatment induced severe liver injury and histopathological abnormalities, which were prevented by SP supplementation. The incidence of liver tumors was significantly reduced from 80 to 20% by SP. Immunohistochemical results indicated that both PCNA and p53 were highly expressed in the liver of DBN-treated rats, but were significantly reduced by SP supplementation. Molecular analysis indicated that SP treatment inhibited cell proliferation, which was accompanied by increased p21 and decreased Rb expression levels at 48hrs post-treatment. In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48hrs. This is the first report of the in vivo chemopreventive effect of SP against DBN-induced rat liver cytotoxicity and carcinogenesis, suggesting its potential use in chemoprevention of cancer.     

Prominent crista terminalis mimicking a right atrial mass: case report

Primary heart neoplasms are rare occurring with an estimated incidence of 0.0017-0.19%. Myxoma is the most prevalent primary heart tumor. The right atrium is an unusual localization, occurring only in 15-20% of myxoma cases. We report a rare case of a massive right atrial myxoma causing tricuspid valve obstruction and presenting as syncope and exertional dyspnea. This case illustrates the influence of myxoma's size, position and mobility as well as patient's body posture and respiration to the development of signs and symptoms. Three-dimensional echocardiography proved useful in surgery planning, allowing a better definition of the tumor outline and attachment.     

Total solid-phase synthesis of bombesin analogs with different functional groups at the C-terminus.

Five bombesin analogs with different functional groups at the C-terminus were synthesized using a solid-phase strategy. The protocols were optimized using 4-(hydroxymethyl)benzoic acid (HMBA) resin to synthesize a common precursor followed by nucleophilic cleavage of the base sensitive peptide ester linkage. The C-terminal modifications included ethylamide, butylamide, methyl ester, propyl ester and hydrazide. Cleavage from the resin was possible with the fully protected or deprotected precursor peptide; however, higher purity of the final products was achieved when cleavage protocols were conducted after side-chain deprotection. The synthesized peptides were analyzed and characterized using reverse phase HPLC and ESI-MS. The peptides were obtained in 13-32% overall recovery, calculated from the coupling efficiency of the first amino acid residue, and in 91-97% purity.     

Endothelium and myocyte cellular insulin receptor alterations in a rat model of myocardial infarction

Ischemic heart disease is considered to be one of the leading causes of death in adults. While extensive research on mechanisms contributing to the pathogenesis of myocardial infarction (MI) has been underway, it is not known whether insulin receptor characteristics and postreceptor signaling have been fully addressed as yet. Present work attempts to investigate whether the remodeling process effectively induces alteration(s) in insulin-binding characteristics at the coronary endothelium and cardiomyocytes using a rat heart model of MI. MI was induced by ligation of the left anterior descending coronary artery of adult male Sprague-Dawley rats. Two animal groups were used in the study: (i) sham-operated CHAPS-untreated and CHAPS-treated, and (ii) MI CHAPS-untreated and MI CHAPS-treated. A physical model describing 1:1 stoichiometry of reversible insulin binding to its receptors present on the endothelium and at cardiomyocytes after CHAPS treatment was considered for data analysis. Quantitation of the collected effluents after heart perfusion, the inlet at the aortic and outlet at the coronary sinus sites, were curve fitted using a first-order Bessel function, which determines the binding constants (k(n)), the reversible constant (k(-n)), the dissociation constant (k(d) = k(-n)/k(n)), and the residency time constant (tau = 1/k(-n)). In addition, hearts were excised, separated into right and left ventricles, and individually weighed, and areas of infarcted regions were measured. Results of the MI group showed significant increases in relative heart mass, left ventricle mass, and right ventricle mass normalized to total body mass. MI induced severe ischemia and irreversible myocardial injury as assessed by planimetry and histologic studies. The data showed differences in insulin receptor affinities at the endothelial and cardiac myocytes in the sham and in the MI-operated rats. The observed reduction in the binding affinity of insulin at the myocyte postinfarction may explain the pathogenic role of insulin in ischemic heart disease and, hence, resistance. Therefore, insulin administration during and post MI might be cardioprotective.     

Ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves

Most estrous cycles in cows consist of 2 or 3 waves of follicular activity. Waves of ovarian follicular development comprise the growth of dominant follicles some of which become ovulatory and the others are anovulatory. Ovarian follicular activity in cows during estrous cycle was studied with a special reference to follicular waves and the circulating concentrations of estradiol and progesterone. Transrectal ultrasound examination was carried out during 14 interovulatory intervals in 7 cows. Ovarian follicular activity was recorded together with assessment of serum estradiol and progesterone concentrations. Three-wave versus two-wave interovulatory intervals was observed in 71.4% of cows. The 3-wave interovulatory intervals differed from 2-wave intervals in: 1) earlier emergence of the dominant follicles, 2) longer in length, and 3) shorter interval from emergence to ovulation. There was a progressive increase in follicular size and estradiol production during growth phase of each wave. A drop in estradiol concentration was observed during the static phase of dominant anovulatory follicles. The size of the ovulatory follicle was always greater and produced higher estradiol compared with the anovulatory follicle. In conclusion, there was a predominance of 3-wave follicular activity that was associated with an increase in length of interovulatory intervals. A dominant anovulatory follicle during its static phase may initiate the emergence of a subsequent wave. Follicular size and estradiol concentration may have an important role in controlling follicular development and in determining whether an estrous cycle will have 2 or 3-waves.     

Effects of lycopene supplementation in patients with localized prostate cancer

Epidemiological studies have shown an inverse association between dietary intake of lycopene and prostate cancer risk. We conducted a clinical trial to investigate the biological and clinical effects of lycopene supplementation in patients with localized prostate cancer. Twenty-six men with newly diagnosed prostate cancer were randomly assigned to receive a tomato oleoresin extract containing 30 mg of lycopene (n = 15) or no supplementation (n = 11) for 3 weeks before radical prostatectomy. Biomarkers of cell proliferation and apoptosis were assessed by Western blot analysis in benign and cancerous prostate tissues. Oxidative stress was assessed by measuring the peripheral blood lymphocyte DNA oxidation product 5-hydroxymethyl-deoxyuridine (5-OH-mdU). Usual dietary intake of nutrients was assessed by a food frequency questionnaire at baseline. Prostatectomy specimens were evaluated for pathologic stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia. Plasma levels of lycopene, insulin-like growth factor-1, insulin-like growth factor binding protein-3, and prostate-specific antigen were measured at baseline and after 3 weeks of supplementation or observation. After intervention, subjects in the intervention group had smaller tumors (80% vs 45%, less than 4 ml), less involvement of surgical margins and/or extra-prostatic tissues with cancer (73% vs 18%, organ-confined disease), and less diffuse involvement of the prostate by high-grade prostatic intraepithelial neoplasia (33% vs 0%, focal involvement) compared with subjects in the control group. Mean plasma prostate-specific antigen levels were lower in the intervention group compared with the control group. This pilot study suggests that lycopene may have beneficial effects in prostate cancer. Larger clinical trials are warranted to investigate the potential preventive and/or therapeutic role of lycopene in prostate cancer.     

Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U-13C8]phenylacetate yielded ring-1,2-dihydroxy-1,2-dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded delta3-dehydroadipate and 3-hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring-13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho-hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl-CoA is attacked by a ring-oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl-CoA, which is not re-oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl-CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent beta-oxidation-type degradation of the resulting CoA dicarboxylate via beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.     

Pre-Breeding Genetic Diversity Assessment of Tomato (<i>Solanum lycopersicum</i> L.) Cultivars Based on Molecular,Morphological and Physicochemical Parameters

Appropriate knowledge of the parental cultivars is a pre-requisite for a successful breeding program. This study characterized fruit yield, quality attributes, and molecular variations of ten tomato cultivars during three consecutive generations under greenhouse conditions. Peto 86, Castle Rock, and Red Star cultivars showed the highest fruit yield (kg/plant), total phenolic compounds (TPC), and sap acidity. Principal component analysis categorized the evaluated fruit yield into three groups based on their quality attributes. A robust positive correlation appeared among traits inside each group. A positive correlation was likewise noticed between the first and the second groups. However, a negative correlation was detected between the first, the second and the third group. Molecular profiling, using seven inter-simple sequence repeat (ISSR) primers, produced 60 loci, including 49 polymorphic loci. The molecular analysis also pinpointed the highest genetic similarity (0.92) between P73 and Moneymaker, while the lowest genetic similarity (0.46) was observed between Castle Rock and Moneymaker. The cultivars P73 and Moneymaker showed the lowest genetic distance (2.24), while the highest genetic distance (5.92) was observed between Super Marmand and Peto86, on the one hand, and between Castle Rock and Moneymaker, on the other hand. The chemical analysis of fruit sap indicated the highest levels of TPC, total flavonoids, anthocyanin, ascorbic acid and total soluble solids in Peto 86 and Castle Rock cultivars. Phylogeny analysis of tomato cultivars based on morphological and molecular attributes indicated four distinct clades. Peto 86, Castle Rock, and Red star cultivars can be recommended for the tomato hybridization breeding programs in the future, with other tomato cultivars as potentially high-yielding parents.