Determination of salicinoids by micro-high-performance liquid chromatography and photodiode array detection.

INTRODUCTION: Plant-derived salicinoids (conjugates of glucose and salicylate phenolic moieties) are potent modulators of plant-herbivore interactions. We demonstrate the use of micro-high-performance liquid chromatography (µHPLC) with photodiode-array detection (DAD) for quantification of four salicinoids (salicin, salicortin, hydroxycyclohexen-on-oyl salicortin and tremulacin) in methanolic extracts of Populus. OBJECTIVE: To develop and implement a solvent-conserving µHPLC method to quantify salicinoids in methanolic extracts of Populus tissue. METHODS: Salicinoids were extracted from Populus tissue into methanol, filtered, and introduced to µHPLC. Extracted analytes were separated on a Zorbax SB C18-column with a binary gradient of methanol and water (with 2% tetrahydrofuran; 20 µL/min), and quantified by DAD (274 nm). We confirmed measurement reliability through standard addition, comparison with an accepted method, and assessment of chromatographic peak purity by ultraviolet absorbance spectra. RESULTS: Method detection and quantification limits for the salicinoids as a percentage of dry leaf weight were as follows: salicin (0.1%, 0.2%), salicortin (0.001%, 0.02%), hydroxycyclohexen-on-oyl salicortin (0.02%, 0.06%) and tremulacin (0.0006%, 0.002%). Calibrations by external standardisation were linear over 1.5 orders of magnitude with acceptable accuracy and reproducibility. CONCLUSION: Micro-HPLC can serve as a solvent-conserving alternative to conventional HPLC for quantification of salicinoids in Populus tissue

Persistence of 2,4-D and its effects on benthic macroinvertebrates following spring treatment of Eurasian Watermilfoil, Myriophyllum spicatum L. in two lakes in southeastern Wisconsin, USA.

The purpose of this study was to determine the persistence of 2,4-dichlorophenoxyacetic acid (2,4-D) applied to two lakes (one mesotrophic and one eutrophic) for the control of Eurasian watermilfoil (EWM), and to determine the impacts of 2,4-D on benthic macroinvertebrates in one of the lakes. One lake was treated with a liquid formulation, and the other with a slow release granular formulation of 2,4-D. Concentrations of 2,4-D in the water column were highest 1 and 2 days post-treatment and declined to below detection limits by 7 and 10 days post-treatment. We observed negative correlations between days post-treatment and taxa richness, and between days post-treatment and abundance of three of 12 taxonomic groups of macroinvertebrates. Lake managers need to balance control of EWM with possible impacts of 2,4-D to nontarget organisms.

The efficacy of St. John's Wort in patients with minor depressive symptoms or dysthymia--a double-blind placebo-controlled study.

INTRODUCTION: We studied the efficacy of St. John's Wort compared with placebo in patients with minor depressive symptoms or dysthymia, with the main focus on which diagnostic entities are optimally amenable to treatment with two different doses of Hypericum, and which are not. METHODS: One hundred and fifty patients, 25-70 years old, meeting ICD-10 criteria for mild or moderately severe depressed episodes or with dysthymia, and having a 17-item Hamilton Depression Scale for Depression (HAM-D) total score between 7 and 17, were randomly assigned to an extract. The extract, PM235, manufactured by Cederroth International AB, Sweden, was given t.i.d. in a lower (0.12% hypericine) or a higher (0.18% hypericine) formulation, based on 270mg extractions or identical placebo. Clinical response was defined by HAM-D as a 50% reduction and/or a score 7. The Beck Depression Inventory (BDI) and Visual Analog Scales (VAS) were used as secondary efficacy parameters. Measures were conducted at screening, baseline, and after 3 and 6 weeks of treatment. RESULTS: We found a large discrepancy in response between dysthymic and non-dysthymics, the latter seemingly more sensitive to Hypericum. HAM-D showed tendency but no significance toward a more frequent improvement of the non-dysthymics treated with Hypericum (p=0.057). BDI criteria showed significance (p=0.045) for both doses of Hypericum compared to placebo. Pooling high- and low-dose groups together, a significant reduction for HAM-D7 and BDI criteria was found among non-dysthymic patients (p=0.03). Significant improvement in response to Hypericum was found in symptoms reflected by VAS - again only in non-dysthymic patients (p=0.041). DISCUSSION: We observed, a tendency toward a more frequent significant improvement of the non-dysthymic patient treated with PM235, though this did not reach the level of statistical significance. In a secondary analysis, pooling both hypericine-treated groups concluded that Hypericum has a clinical significant effect in minor depressed patients with HAM-D up to 17. This finding was significant only in non-dysthymic patients.

Femal, a herbal remedy made from pollen extracts, reduces hot flushes and improves quality of life in menopausal women: a randomized, placebo-controlled, parallel study.

BACKGROUND: The fact that hormone replacement therapy has been claimed to increase the risk of breast cancer has made it relevant to search for new non-hormonal treatments of menopausal symptoms. OBJECTIVES: This study aimed to evaluate whether Femal, a herbal remedy made from pollen extracts, alleviates the symptoms of the menopause, especially hot flushes. DESIGN: A randomized, double-blind, placebo-controlled, parallel trial of 64 menopausal women, of whom 54 completed the trial. After an initial run-in phase of 1 month, the women were randomly given either two Femal tablets each morning, or two identical placebo tablets, for 3 months of treatment. On inclusion, and then at 4-week intervals, the patients were asked to evaluate 16 symptoms of the menopause using Menopause Rating Scales (MRS). In addition, every day throughout the study, certain menopausal symptoms were recorded in a diary. RESULTS: The two treatment groups were identical regarding demographic data and the initial symptom scores. In the active-treatment group, 65% responded with a reduction in hot flushes compared with 38% in the placebo group (p<0.006) and, in this group, the number of hot flushes registered in diaries declined after 3 months by 27% as compared to the placebo group (p<0.026). MRS evaluation of hot flushes yielded similar results (p<0.031). There were 23% and 22% decreases in hot flushes after 2 and 3 months of treatment, respectively, and after both intervals of time the inter-group comparisons were significantly affected. An overall evaluation of the trend in 15 other 'quality-of-life' parameters showed likewise in favor of the pollen extract (p<0.031). CONCLUSION: The pollen extract Femal significantly reduces hot flushes and certain other menopausal symptoms when compared to placebo.

Parasomnias decline during pregnancy.

OBJECTIVES: A survey of the effects of pregnancy on parasomnias. MATERIAL AND METHODS: In an area of a central hospital and the maternity care units in the nearby rural community, women were interviewed during and after their pregnancy with a series of five questionnaires to assess the frequency of their parasomnias. The first questionnaire covered the 3 months before becoming pregnant, the next three the trimesters of pregnancy and the last one the 3 months after delivery. Altogether 325 mothers filled all the five questionnaires and constitute the study group. RESULTS: The total number of parasomnias declined (P < 0.001) during pregnancy and even more among the primiparas than among the multiparas (difference until third trimester, P=0.02). Among various parasomnias reported, sleep talking and sleepwalking decreased from the prepregnant period to the second trimester (22.8 vs 12.6%, change P=0.003), and the reported sleep starts also diminished from the prepregnant time to the first trimester (78.5 vs 63.1%, P < 0.001), but these phenomena did not change further during the follow-up. Altogether 55.7% of the women reported having nightmares 3 months before the pregnancy, and 47.7, 49.5, 41.2 and 40.3% (change from the prepregnant period, P < 0.001), respectively, at first, second and third trimester and after the delivery. Reported hypnagogic hallucinations decreased from the prepregnant time to the first trimester (9.8 vs 6.5%, P=0.027), but returned thereafter to the previous level. During the prepregnant period, 25.8% of the women reported bruxism and only 19.9% during the first trimester (P=0.009). Though the prevalence of sleep paralysis decreased during the first trimester of pregnancy, it was the only parasomnia that increased during later pregnancy (from 5.7 to 13.3% in the second trimester, P < 0.013). CONCLUSIONS: The reported frequency of most parasomnias decreases during pregnancy and even more in primiparas than multiparas.

Effects of pregnancy on mothers' sleep.

OBJECTIVES: To survey the effects of pregnancy on mothers' sleep. METHODS: Mothers were interviewed during and after pregnancy with a series of five questionnaires to assess alterations in their sleep. The first questionnaire covered the 3 months before becoming pregnant, the next three the trimesters of pregnancy and the last the 3 months after delivery. The study was carried out in a central hospital and the maternity care units in the nearby rural community. Altogether, 325 pregnant women completed all five questionnaires. RESULTS: The total amounts of reported sleep and of nocturnal sleep increased significantly during the first trimester of pregnancy, began to decrease thereafter and were shortest during the 3 months after pregnancy. During late pregnancy expectant mothers over 30 years of age reported less sleep than those under 30. During the whole pregnancy, but increasingly toward the end of pregnancy, sleep became more restless and fragmentary and its subjective quality worsened, due at least partly to increased restless legs and nightly awakenings increasing with advancing pregnancy. CONCLUSIONS: The subjective quality of sleep is disturbed as early as the first trimester of pregnancy, although total sleeping time increases. The amount of reported sleep begins to decrease in the second trimester. The frequency of reported sleep disturbances, such as restless legs syndrome and nocturnal awakenings, is maximum in the third trimester but is about normal within 3 months after delivery.

Treatment with insulin lispro changes the insulin profile but does not affect the plasma concentrations of IGF-I and IGFBP-1 in type 1 diabetes.

OBJECTIVE: IGF-I levels in patients with type 1 diabetes without endogenous insulin production are low. Our aim was to examine whether the plasma insulin profile obtained by treatment with the insulin analogue lispro has a different effect on plasma concentrations of IGF-I and IGFBP-1 than that seen during treatment with conventional human insulin (regular insulin). DESIGN AND PATIENTS: Twelve patients with type 1 diabetes, age 47.8 +/- 2.4 years (mean +/- SEM), body mass index 26.5 +/- 1.0 kg/m2, diabetes duration 30.5 +/- 3.2 years participated in this open label randomized cross-over study. IGF-I and IGFBP-1 levels were measured at the end of 6 weeks treatment with each insulin being administered by a continuous subcutaneous insulin infusion. IGF-I was measured fasting while IGFBP-1, free insulin and blood glucose were measured fasting and repeatedly after a morning meal preceded by an insulin bolus dose. RESULTS: Lispro gave a marked insulin peak of 135 +/- 20 pmol/l 50 minutes after injection. After an initial rapid rise, human regular insulin reached a plateau of approximately 50 pmol/l. The plasma free insulin area under the curve (AUC) from 0710 h to 0910 h was more than twice as large on lispro as on regular insulin (P = 0.01). Plasma IGF-I concentration was 78.8 +/- 10.9 microg/l on lispro and 82.3 +/- 10.5 microg/l on human regular insulin (not significant). AUC for IGFBP-1 did not show a significant difference even when divided from 0710 h to 0910 h and from 0930 h to 1430 h. Blood glucose AUC after administration of the bolus was significantly lower during treatment with lispro (P = 0.006) but glycosylated haemoglobin (HbA1c) was 6.4 +/- 0.2% on both therapies. CONCLUSIONS: Our results indicate that the effect of lispro on IGF-I and IGFBP-1 in patients with type 1 diabetes does not differ from that of human regular insulin.

Modified-release formulation of tizanidine in chronic tension-type headache.

The efficacy of the modified-release formulation of tizanidine (Sirdalud) was compared with placebo in a randomized, double-blind, parallel-group study of 138 women and 47 men, aged 18 to 79 years, with a history of chronic tension-type headache (IHS categories 2.2 and 2.3). The treatment period was 6 weeks preceded by a 2-week prerandomization period. The patients were randomly assigned to receive 6-mg Sirdalud, 12-mg Sirdalud MR, or placebo. The study medication was taken once per day, orally in the evening. Efficacy was measured by visual analog scale, the number of headache-free days, the daily duration of headache, and the use of paracetamol. The primary end point was the severity of daily headache derived from visual analog scale scores covering the last 2 treatment weeks. One hundred sixty patients (56 in the 6-mg group, 49 in the 12-mg group, and 55 in the placebo group) completed the study. The severity of the headache decreased similarly in the treatment groups and the placebo group. The visual analog scale values decreased from the prerandomization values by 53% in the 6-mg group, 48% in the 12-mg group, and 52% in the placebo group. The modified-release formulation of tizanidine in doses up to 12 mg taken in the evening is not superior to placebo in the treatment of chronic tension-type headache. The placebo effect was unexpectedly strong in the present study, supporting the view that psychophysiological mechanisms are of considerable importance in sustaining chronic tension-type headache.

Postoperative analgesia by D-myo-inositol-1,2,6-trisphosphate in patients undergoing cholecystectomy.

UNLABELLED: D-myo-inositol-1,2,6-trisphosphate (1,2,6-IP3) possesses antiinflammatory properties, such as reduced eicosanoid synthesis and inhibition of inflammation-induced edema. These properties suggest possible analgesic effects. The analgesic effect of 1,2,6-IP3 was evaluated in a double-blind, randomized study in 24 patients undergoing cholecystectomy. Ten patients received 1,2,6-IP3 as an intravenous (i.v.) bolus dose of 240 mg, followed by a continuous i.v. infusion at 90 mg/h for 24 h. The placebo group (n = 14) received corresponding volumes of isotonic saline. Postoperative pain (visual analog pain scale; VAS) and opiate analgesic requirements (ketobemidon) were evaluated during five postoperative days. Results showed significantly reduced pain during the first five postoperative days in patients treated with 1,2,6-IP3, as measured by using a VAS (P < 0.05). The requirements of opioid analgesics were significantly reduced during the first three postoperative days (P < 0.05). No drug-related side effects were observed. Results of the present study demonstrate a potent and long-lasting analgesic effect of 1,2,6-IP3, possibly related to its antiinflammatory properties. IMPLICATIONS: A new antiinflammatory drug under investigation, inositol-1,2,6-trisphosphate, was evaluated as a possible analgesic in a pilot study during the postoperative period in cholecystectomized patients. Results showed significantly lower pain assessment and opioid consumption, which should encourage further studies.

Thrombolysis for acute ischemic stroke in the primary antiphospholipid syndrome.

Successful intravenous thrombolytic treatment for acute major ischemic stroke was given to a 24-year-old woman with primary antiphospholipid syndrome. Our patient was a smoker and used oral contraceptives containing estrogen at the time of the stroke. We discuss risk factors for stroke and the treatment of arterial thrombosis in patients with antiphospholipid antibodies.

Plasmid and chromosomal DNA recovery by electroextraction of cyanobacteria.

High voltage electroporation has been investigated as a method for rapid recovery of plasmid and chromosomal DNA from the cyanobacteria Nostoc PCC 7121, Synechococcus PCC 7002, and Anabaena PCC 7120. Pulses of 18 kV/cm and higher applied to concentrated Nostoc cells carrying a shuttle plasmid (pRL25) resulted in copious release of nucleic acids and phycobiliproteins into the suspending medium. Small portions of these supernatants, when electroporated with Escherichia coli, gave rise to hundreds of E. coli transformants which contained pRL25. Electroporation of Synechococcus carrying plasmid pAQE19 did not cause detectable release of macromolecules but did reveal a low-level, voltage independent 'leakage' of pAQE19 into the medium. Electroextraction of Nostoc or Anabaena followed by addition of E. coli and delivery of a second high-voltage pulse permitted direct, one-cuvette transfer of shuttle plasmids from these cyanobacteria into E. coli. Electroextraction of single cyanobacterial colonies, as shown for Nostoc, also released sufficient chromosomal DNA for amplification of specific sequences by the polymerase chain reaction.

Platelet P1, P4-Di (adenosine-51) tetraphosphate (AP4A) in migraine patients before and during beta-adrenoceptor blockade.

P1,P4-Di(adenosine-51) tetraphosphate (AP4A) is a metabolically inactive nucleotide which can be released from platelet dense granules. This study was designed firstly, to investigate whether platelet content of AP4A was decreased in patients with classical migraine and secondly, whether the content of AP4A was changed by beta-adrenoceptor blockade. No significant difference in platelet dense granule content of AP4A, was observed between 10 migraine patients and 10 normal controls. Both metoprolol, a beta 1-selective blocker and propranolol, a non-selective beta-blocker significantly decreased the migraine attack rate. However, while propranolol significantly reduced the platelet content of AP4A, metoprolol did not. Therefore, the present data suggest that platelet dense granule release, as estimated by the content of AP4A is not of major importance in migraine.

Influence of metoprolol CR/ZOK on plasminogen activator inhibitor (PAI-1) in man: a pilot study.

Recent primary and secondary preventive trials have shown that long-term metoprolol therapy reduces the risk of acute cardiovascular complications. To test whether part of this beneficial long-term effect may be due to effects on the fibrinolytic system, three pilot studies were performed; two in healthy individuals, and one in patients with mild hypertension or uncomplicated atrial fibrillation. The effect of metoprolol CR/ZOK (controlled release) 100-200 mg daily, on plasminogen activator inhibitor activity (PAI-1) in plasma was measured. In addition serum triglycerides and orosomucoid were analyzed. All the individuals were included in double-blind placebo controlled cross-over trials with treatment periods ranging from 4 days to 3 weeks. During metoprolol therapy PAI-1 values were reduced, while orosomucoid and triglyceride levels were unchanged. A linear inverse correlation was found between fibrinolysis and PAI-1 activity in plasma, indicating that PAI-1 activity serves as an indicator of fibrinolysis. PAI-1 activity and triglycerides were significantly correlated during placebo and metoprolol therapy. In conclusion, our results in these pilot studies suggest that metoprolol enhances fibrinolytic activity as seen by reduced PAI-1 activity. These results should be further confirmed and put into relation of clinical effects of the therapy.