Nonadherence by Serum Drug Analyses in Resistant Hypertension: 7-Year Follow-Up of Patients Considered Adherent by Directly Observed Therapy.

Background Measurement of serum concentrations of drugs is a novelty found useful in detecting poor drug adherence in patients taking ≥2 antihypertensive agents. Regarding patients with treatment-resistant hypertension, we previously based our assessment on directly observed therapy. The present study aimed to investigate whether serum drug measurements in patients with resistant hypertension offer additional information regarding drug adherence, beyond that of initial assessment with directly observed therapy. Methods and Results Nineteen patients assumed to have true treatment-resistant hypertension and adherence to antihypertensive drugs based on directly observed therapy were investigated repeatedly through 7 years. Serum concentrations of antihypertensive drugs were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry from blood samples taken at baseline, 6-month, 3-year, and 7-year visits. Cytochrome P450 polymorphisms, self-reported adherence and beliefs about medicine were performed as supplement investigations. Seven patients (37%) were redefined as nonadherent based on their serum concentrations during follow-up. All patients reported high adherence to medications. Nonadherent patients expressed lower necessity and higher concerns regarding intake of antihypertensive medication (P=0.003). Cytochrome P450 polymorphisms affecting metabolism of antihypertensive drugs were found in 16 patients (84%), 21% were poor metabolizers, and none were ultra-rapid metabolizers. Six of 7 patients redefined as nonadherent had cytochrome P450 polymorphisms, however, not explaining the low serum drug concentrations measured in these patients. Conclusions Our data suggest that repeated measurements of serum concentrations of antihypertensive drugs revealed nonadherence in one-third of patients previously evaluated as adherent and treatment resistant by directly observed therapy, thereby improving the accuracy of adherence evaluation. Registration URL: https://www.clinicaltrials.gov; unique identifier: NCT01673516.

Development of UHPLC-MS/MS methods to quantify 25 antihypertensive drugs in serum in a cohort of patients treated for hypertension.

We developed three ultra-high pressure liquid chromatography coupled to mass spectrometry detection (UHPLC-MS/MS) methods to quantify 25 antihypertensive drugs in serum samples. Patient-reported drug lists were collected, and drug concentrations were analysed in samples from 547 patients, half with uncontrolled hypertension, and all treated with ≥ 2 antihypertensive drugs. For sample preparation, serum was mixed with deuterated internal standards and acetonitrile and precipitated. Aliquots of the supernatant were injected on UHPLC-MSMS with a C18 reversed phase column. The mobile phase was 0.1 % HCOOH (formic acid) in water and 0.1 % HCOOH in acetonitrile (except in methanol for spironolactone/canrenone) at a flow rate of 0.4 mL/min. The calibrators and internal controls were prepared in Autonorm™. The calibration ranges were wide, and the models were linear or quadratic with squared correlation coefficients ≥ 0.97. The limits of detection and quantification, specificity, carry-over, and matrix effects were acceptable. The accuracy of the internal controls was in the range 85-121 %, and the intermediate precision for all drugs was 4-28 %. The patient-reported antihypertensive drug use and the detected serum drug concentrations were in accordance with that most frequently prescribed nationally. The percent non-detectable level was 5-10 % for bendroflumethiazide, doxazosin, nifedipine, and ramipril. Often the drug dose chosen was lower than the recommended maximum daily dose. We report the maximum (Cmax) and minimum (Cmin) drug concentrations after drug intake. The inter-individual pharmacokinetic variability at Cmin was 18-fold for hydrochlorothiazide, 22-fold for losartan carboxyl acid, 26-fold for amlodipine, 44-fold for candesartan, and 50-fold for valsartan. Our methods are suitable for measuring antihypertensive drugs in patient serum for therapy control.

Toxigenic Fusarium spp. as determinants of trichothecene mycotoxins in settled grain dust.

Trichothecenes are immunosuppressive mycotoxins produced mainly by Fusarium spp. and often are detected as natural contaminants of grain and other agricultural products. Exposure to trichothecenes through inhalation during grain work may represent possible health risks for grain farmers. We aimed, therefore, to investigate the level of Fusarium spp. and trichothecenes in settled grain dust collected during work on 92 Norwegian farms. Mycotoxins were determined by gas chromatography-mass spectrometry, whereas the Fusarium spp. were identified and quantified both by species-specific semiquantitative polymerase chain reaction (PCR) and by cultivation. All potential trichothecene-producing molds in the grain dust were quantified using a PCR assay specific for tri5, the gene coding for trichodiene synthase that catalyzes the first step in the trichothecene biosynthesis. We performed correlation analysis between mold-DNA and mycotoxins to assess whether the PCR-detected DNA could be used as indicators of the mycotoxins. The methodological problem of detecting small amounts of airborne mycotoxins during grain work may then be avoided. Whereas the trichothecene-producing Fusarium species in grain dust could not be identified or quantified to a sufficient extent by cultivation, all investigated Fusarium spp. could be specifically detected by PCR and quantified from the DNA agarose gel band intensities. Furthermore, we observed a strong correlation between the trichothecenes HT-2 toxin (HT-2) or T-2 toxin (T-2) and DNA specific for tri5 (r = 0.68 for HT-2 and r = 0.50 for T-2; p < 0.001), F. langsethiae (r = 0.77 for HT-2 and r = 0.59 for T-2; p < 0.001), or F. poae (r = 0.41 for HT-2 and r = 0.35 for T-2; p < 0.001). However, only a moderate correlation was observed between the trichothecene deoxynivalenol (DON) and the combination of its producers, F. culmorum and F. graminearum (r = 0.24, p = 0.02), and no significant correlation was observed between DON and tri5. PCR clearly improved the detection of toxigenic Fusaria as potential sources of health risks for farmers inhaling grain dust during work, but the use of Fusarium-DNA as indicators for trichothecenes should be used cautiously.

Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides.

The production of mycotoxins and other metabolites by 109 strains of Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, and F. kyushuense was investigated independently in four laboratories by liquid or gas chromatography analyses of cultural extracts with UV diode array, electron capture, or mass spectrometric detection systems. From the compiled results, it was found that F. langsethiae consistently produced the trichothecenes diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2), and neosolaniol (NEO) and, to a lesser extent, some additional trichothecene derivatives. F. langsethiae also produced culmorins, chrysogine (CHRYS), aurofusarin (AUF), and enniatin (EN). F. sporotrichioides showed a metabolite profile similar to that of F. langsethiae, while F. poae had a different profile as 41 of 49 strains produced nivalenol (NIV) and other 8-keto trichothecenes, in addition to DAS and derivatives of this metabolite. Only a trace amount of NIV was detected from one strain of F. kyushuense. In summary, all the three core taxa of this joint study were found to produce trichothecenes. Fusarin C (F-C) was not detected from F. langsethiae, but it was produced by F. poae and F. sporotrichioides. Aurofusarin was only detected from a few strains of F. langsethiae, while nearly all strains of F. poae and F. sporotrichioides produced this compound. In contrast, chrysogine was not detected from F. poae, but was produced by the other two taxa. Production of enniatins was scattered among the three main taxa of this study, whereas beauvericin (BEA) was produced by many strains of F. poae and F. sporotrichioides. Only one odd strain of F. langsethiae (IBT 9959) produced beauvericin. However, the status of this strain is uncertain. By a polyphasic approach using species-specific metabolite profiles, the fruity odour of F. poae, and morphological observations, it was concluded that F. langsethiae, F. poae, and F. sporotrichioides should be regarded as three significant taxa at a species level.

Trichothecene mycotoxins and their determinants in settled dust related to grain production.

We hypothesise that inhalant exposure to mycotoxins causes developmental outcomes and certain hormone-related cancers that are associated with grain farming in an epidemiological study. The aim of the present study was to identify and validate determinants of measured trichothecene mycotoxins in grain dust as work environmental trichothecene exposure indicators. Settled grain dust was collected in 92 Norwegian farms during seasons of 1999 and 2000. Production characteristics and climatic data were studied as determinants of trichothecenes in settled dust samples obtained during the production of barley (N = 59), oats (N = 32), and spring wheat (N = 13). Median concentrations of trichothecenes in grain dust were <20, 54, and < 50 mg/kg (ranges < 20-340, < 30-2400, and < 50-1200) for deoxynivalenol (DON), HT-2 toxin (HT-2) and T-2 toxin (T-2) respectively. Late blight potato rot (fungal) forecasts have been broadcast in Norway to help prevent this potato disease. Fungal forecasts representing wet, temperate, and humid meteorological conditions were identified as strong determinants of trichothecene mycotoxins in settled grain dust in this study. Differences in cereal species, production properties and districts contributed less to explain mycotoxin concentrations. Fungal forecasts are validated as indicators of mycotoxin exposure of grain farmers and their use in epidemiological studies may be warranted.