Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Sea lice as a density-dependent constraint to salmonid farming

Fisheries catches worldwide have shown no increase over the last two decades, while aquaculture has been booming. To cover the demand for fish in the growing human population, continued high growth rates in aquaculture are needed. A potential constraint to such growth is infectious diseases, as disease transmission rates are expected to increase with increasing densities of farmed fish. Using an extensive dataset from all farms growing salmonids along the Norwegian coast, we document that densities of farmed salmonids surrounding individual farms have a strong effect on farm levels of parasitic sea lice and efforts to control sea lice infections. Furthermore, increased intervention efforts have been unsuccessful in controlling elevated infection levels in high salmonid density areas in 2009-2010. Our results emphasize host density effects of farmed salmonids on the population dynamics of sea lice and suggest that parasitic sea lice represent a potent negative feedback mechanism that may limit sustainable spatial densities of farmed salmonids.     

Benefit from B-lymphocyte depletion using the anti-CD20 antibody rituximab in chronic fatigue syndrome. A double-blind and placebo-controlled study

Background

Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during cancer chemotherapy in a patient with synchronous CFS and lymphoma spurred a pilot study of B-lymphocyte depletion using the anti-CD20 antibody Rituximab, which demonstrated significant clinical response in three CFS patients.

Methods and Findings

In this double-blind, placebo-controlled phase II study ({"type":"clinical-trial","attrs":{"text":"NCT00848692","term_id":"NCT00848692"}}NCT00848692), 30 CFS patients were randomised to either Rituximab 500 mg/m² or saline, given twice two weeks apart, with follow-up for 12 months. Xenotropic murine leukemia virus-related virus (XMRV) was not detected in any of the patients.The responses generally affected all CFS symptoms. Major or moderate overall response, defined as lasting improvements in self-reported Fatigue score during follow-up, was seen in 10 out of 15 patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo group (p = 0.003). Mean response duration within the follow-up period for the 10 responders to Rituximab was 25 weeks (range 8–44). Four Rituximab patients had clinical response durations past the study period. General linear models for repeated measures of Fatigue scores during follow-up showed a significant interaction between time and intervention group (p = 0.018 for self-reported, and p = 0.024 for physician-assessed), with differences between the Rituximab and Placebo groups between 6–10 months after intervention. The primary end-point, defined as effect on self-reported Fatigue score 3 months after intervention, was negative. There were no serious adverse events. Two patients in the Rituximab group with pre-existing psoriasis experienced moderate psoriasis worsening.

Conclusion

The delayed responses starting from 2–7 months after Rituximab treatment, in spite of rapid B-cell depletion, suggests that CFS is an autoimmune disease and may be consistent with the gradual elimination of autoantibodies preceding clinical responses. The present findings will impact future research efforts in CFS.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00848692","term_id":"NCT00848692"}}NCT00848692     

Decreased heart rate variability in HIV positive patients receiving antiretroviral therapy: importance of blood glucose and cholesterol

The presence of autonomic dysfunction in HIV patients is largely unknown. Early studies found autonomic dysfunction in patients with AIDS. Antiretroviral combination therapy (ART) has dramatically changed the course of the disease and improved prognosis and decreased morbidity.

Aim

To evaluate whether autonomic dysfunction is present in an ART treated HIV population and if so to identify factors of importance.

Methods

HIV patients receiving ART for at least 12 months (n = 97) and an age-matched control group of healthy volunteers (n = 52) were included. All were non-diabetic and had never received medication for hypertension. Following a 10 min resting period a 15 min ECG recording was performed. Heart-rate variability (HRV) analysis was performed in accordance with current guidelines and data reported as mean [interquartile range].

Results

Mean normal-to-normal (NN) and total HRV measured as standard deviation of normal-to-normal (SDNN) was lower in HIV patients compared to controls (905 vs. 982 ms; p<0.001 and 48 vs. 54 ms; p = 0.028, respectively). No differences were found between the groups in parasympathetic activity measured as square root of the mean squared difference of successive NN-intervals (RMSSD) or the percent of differences between adjacent NN intervals greater than 50 ms (pNN50). In the HIV positives, haemoglobin A1c correlated inversely with SDNN, RMSSD and pNN50 (p<0.05). Total cholesterol and LDL-C correlated inversely with RMSSD and pNN50 (p<0.05). Neither HIV duration, HIV-RNA, CD4 cell count nor CD4 nadir correlated with time or phase domain HRV variables.

Conclusions

Moderate autonomic dysfunction is present in HIV positives patients even with suppressed viral load due to ART. The dysfunction is correlated with HbA1c and hypercholesterolemia but not to duration of HIV or whether the patients were receiving protease inhibitors as part of the ART regime.     

Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.     

Study protocol: Brief intervention for medication overuse headache - A double-blinded cluster randomised parallel controlled trial in primary care

ABSTRACT: BACKGROUND: Chronic headache (headache [GREATER-THAN OR EQUAL TO] 15 days/month for at least 3 months) affects 2--5% of the general population. Medication overuse contributes to the problem. Medication-overuse headache (MOH) can be identified by using the Severity of Dependence Scale (SDS). A "brief intervention" scheme (BI) has previously been used for detoxification from drug and alcohol overuse in other settings. Short, unstructured, individualised simple information may also be enough to detoxify a large portion of those with MOH. We have adapted the structured (BI) scheme to be used for MOH in primary care. METHODS: A double-blinded cluster randomised parallel controlled trial (RCT) of BI vs. business as usual. Intervention will be performed in primary care by GPs trained in BI. Patients with MOH will be identified through a simple screening questionnaire sent to patients on the GPs lists. The BI method involves an approach for identifying patients with high likelihood of MOH using simple questions about headache frequency and the SDS score. Feedback is given to the individual patient on his/her score and consequences this might have regarding the individual risk of medication overuse contributing to their headache. Finally, advice is given regarding measures to be taken, how the patient should proceed and the possible gains for the patient. The participating patients complete a headache diary and receive a clinical interview and neurological examination by a GP experienced in headache diagnostics three months after the intervention. Primary outcomes are number of headache days and number of medication days per month at 3 months. Secondary outcomes include proportions with 25 and 50% improvement at 3 months and maintenance of improvement and quality of life after 12 months. DISCUSSION: There is a need for evidence-based and cost-effective strategies for treatment of MOH but so far no consensus has been reached regarding an optimal medication withdrawal method. To our knowledge this is the first RCT of structured non-pharmacological MOH treatment in primary care. Results may hold the potential of offering an instrument for treating MOH patients in the general population by GPs. Trial registration ClinicalTrials.gov identifier: NCT01314768.     

Reaction wood – a key cause of variation in cell wall recalcitrance in willow

Background

The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.

Results

A pot trial was designed to test if the ‘RW response’ varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.

Conclusions

Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a ‘RW response’) indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.