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.     

A simulation study of cellular hypertrophy and connexin lateralization in cardiac tissue

Many cardiac diseases coincide with changes in cell size and shape. One example of such a disease is cardiac hypertrophy. It is established that cardiac impulse propagation depends on the cell size, as well as other factors, but interrelations between conduction velocity (CV), cell size, and gap junction (GJ) conductance (g_GJ) are complex. Furthermore, cardiac diseases are often accompanied by connexin (Cx) lateralization. To analyze the effects of cell size and Cx lateralization in cardiac disease, a two-dimensional computer simulation of ventricular myocytes based on the Luo-Rudy model was used. Control cells (80 μm/20 μm (length/diameter)), long cells (160 μm/20 μm), and wide cells (80 μm/40 μm) were simulated as was a redistribution of lateral GJs (constant lateral g_GJ and increased lateral g_GJ). CV in long cells showed high stability, i.e., it declined very slowly when g_GJ was gradually reduced. Wide cells, however, were more affected by reduced g_GJ, resulting in early transition to discontinuous propagation and low CV. Conduction block occurred earlier in enlarged cells than in control cells due to increased cell capacitance. Increased lateral g_GJ stabilized longitudinal CV, which was a result of two-dimensional effects during planar wave propagation. Therefore, Cx lateralization may compensate for cardiac inhomogeneities. High lateral g_GJ and enhanced cell diameter increased the susceptibility to conduction block at tissue expansion, providing a substrate for arrhythmia.     

Antihypertensive and cardioprotective effects of different monotherapies and combination therapies in young spontaneously hypertensive rats – A pilot study

Spontaneously hypertensive rats (SHR) are an established animal model for antihypertensive treatment. The aim of this pilot study was a systematic search for two lines of antihypertensive treatment – a monotherapy and a combination of two drugs – to be applied in a future study on old SHR. Originally, representatives of three drug classes recommended for antihypertensive therapy in humans should be applied, namely captopril (CAP) as an antagonist of the renin-angiotensin-aldosterone system, nifedipine (NIF) as calcium channel blocker and propranolol (PROP) as β-adrenergic blocker. As we observed that PROP had been poorly ingested, all groups with PROP therapy were excluded from the study. CAP (60 mg kg⁻¹ d^-1), NIF (10 mg kg⁻¹ d^-1) or both were administered orally to seven-week-old SHR over 3 weeks. A further group of SHR received no treatment (SHR/CTRL). Age-matched normotensive Wistar-Kyoto rats served as normotensive controls. We examined the effect of the antihypertensive therapies on systolic blood pressure, heart weight and on histological and biochemical markers of cardiac hypertrophy and fibrosis.CAP proved to be the most effective treatment reducing blood pressure and relative heart weight significantly compared to SHR/CTRL without reaching normotensive values. Beginning cardiac fibrosis observed in SHR/CTRL was completely abrogated with CAP treatment. Similar effects were achieved with a combination of CAP and NIF. CAP as monotherapy and CAP + NIF as combination therapy were chosen for the forthcoming study on old SHR.Keyword: Young SHR, Antihypertensive therapy, Blood pressure monitoring, Cardiac hypertrophy, Cardiac fibrosis     

Divergent structure and function of the bicoid gene in Muscoidea fly species

SUMMARY We have investigated the evolution of the bicoid ( bcd ) gene in fly species of the Muscoidea Superfamily. We obtained the complete bcd sequence from the housefly Musca domestica and found polymorphism in the coding region among Musca strains. In addition to Musca , we cloned most of the bcd coding sequences from two blowfly species Calliphora vicina and Lucilia sericata . The 5' and 3' regulatory regions flanking the Musca bcd gene are widely diverged in sequence from Drosophila; however, some important sequence motifs identified in Drosophila bcd are present. The predicted RNA secondary structures of the 3' UTRs are similar, despite sequence divergence. Comparison of Bicoid (Bcd) proteins shows a serine-rich domain of unknown function is present in the Muscoidea species, but is absent in other species. The in vivo function of bcd in Musca was tested by RNAi to mimic loss of function phenotype. We obtained a head defect phenotype similar to weak bcd alleles of Drosophila . Although our comparisons initially suggest functional conservation between species, closer inspection reveals significant differences. Divergence of structural motifs, such as regulatory elements in flanking regions and conservation of protein domains in some species but not in others, points to functional divergence between species. We suggest that the larger embryonic size in Muscoidea species restricts the morphogenetic activity of a weak Bcd activator, which has evolved a more specialized role in head determination and lost some functions in thoracic development.     

Adaptive Packet-size Control for Improved Throughput in Dynamic Access Networks

Cluster Computing - Cognitive radio (CR) is a new intelligent wireless technology that aims at improving spectrum utilization by allowing opportunistic access to the underutilized licensed...     

Effects of introgression of two QTL for fusarium head blight resistance from Asian spring wheat by marker-assisted backcrossing into European winter wheat on fusarium head blight resistance,yield and quality traits

Breeding for fusarium head blight (FHB) resistance of wheat is a continuous challenge for plant breeders. Resistance to FHB is a quantitative trait, governed by several to many genes and modulated by environmental conditions. The presented study was undertaken to assess the effect on improving FHB resistance and on possible unwanted side effects (‘linkage drag’) of two resistance QTL, namely Fhb1 and Qfhs.ifa-5A, from the spring wheat line CM-82036 when transferred by marker-assisted backcrossing into several European winter wheat lines. To achieve these goals, we developed and evaluated fifteen backcross-two–derived families based on nine European winter wheat varieties as recipients and the FHB resistant variety CM-82036 as resistance donor. The QTL Qfhs.ifa-5A had a relatively small impact on increasing FHB resistance. On average lines with Fhb1 plus Qfhs.ifa-5A combined were only slightly more resistant compared to lines with Fhb1 alone. The obtained results suggest that the effect of the spring wheat–derived QTL on improving FHB resistance increases in the order Qfhs.ifa-5A < Fhb1 ≤ Qfhs.ifa-5A plus Fhb1 combined. The genetic background of the recipient line had a large impact on the resistance level of the obtained lines. No systematic negative effect of the spring wheat–derived QTL on grain yield, thousand grain weight, hectoliter weight and protein content was found. The use of spring wheat–derived FHB resistance QTL for breeding high yielding cultivars with improved FHB resistance appears therefore highly promising.     

Pharmacology of gap junctions. New pharmacological targets for treatment of arrhythmia, seizure and cancer?

Intercellular communication in many organs is maintained via intercellular gap junction channels composed of connexins, a large protein family with a number of isoforms. This gap junction intercellular communication (GJIC) allows the propagation of action potentials (e.g., in brain, heart), and the transfer of small molecules which may regulate cell growth, differentiation and function. The latter has been shown to be involved in cancer growth: reduced GJIC often is associated with increased tumor growth or with de-differentiation processes. Disturbances of GJIC in the heart can cause arrhythmia, while in brain electrical activity during seizures seems to be propagated via gap junction channels. Many diseases or pathophysiological conditions seem to be associated with alterations of gap junction protein expression. Thus, depending on the target disease opening or closure of gap junctions may be of interest, or alteration of connexin expression. GJIC can be affected acutely by changing gap junction conductance or--more chronic--by altering connexin expression and membrane localisation. This review gives an overview on drugs affecting GJIC.     

Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure

Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.