V. Blaha

Dopamine and serotonin VMN release is related to feeding status in obese and lean Zucker rats.

Study of neurotransmitter role in food intake regulation in a leptin signaling deficient model, such as the Zucker rat, would benefit in the understanding of mechanisms of human obesity, in which leptin resistance is a common syndrome. We studied dopamine (DA) and serotonin (5-HT) concentrations in vivo in the ventromedial nucleus (VMN) of the hypothalamus, as they relate to eating after food deprivation in obese and lean 9-week-old male Zucker rats. DA and 5-HT concentrations were measured by HPLC via microdialysis before and during refeeding in 24-h food-deprived rats. Before food was provided, mean baseline DA and 5-HT levels were lower in obese than in lean rats (9.2 ± 0.9 vs 15.1 ± 1.9 pg/10 μl, p < 0.01, and 0.68 ± 0.05 vs 1.17 ± 0.02 pg/10 μl, p < 0.001, respectively). Food intake was accompanied by a decrease in DA levels in both obese and lean rats to 64% (p < 0.01) and 65% (p < 0.02) of their baseline levels respectively. 5-HT levels were significantly increased during eating by 41% in obese and 35% in lean rats (p < 0.01) from the baseline levels. Thus in obese rats with altered leptin signaling we found an unaltered pattern of DA and 5-HT release associated with food deprivation and refeeding, but with presence of their low levels. This points to an impaired postsynaptic monoaminergic action to produce an adequate metabolic response in obese Zucker rats in response to feeding state.

Effect of estradiol and progesterone on daily rhythm in food intake and feeding patterns in Fischer rats

The product of meal number x meal size, over time, is food intake. Because estrogens modulate feeding activity via their action on the hypothalamus, and because there is a diurnal rhythm in the expression of cytoplasmic estrogen receptors and in estrogen binding activity, the present study examined the effects of ovariectomy and later hormone therapy on acute changes in body weight, and on the meal number-to-meal size relationship as reflected by food intake in the dark/light feeding patterns, in adult female rats in the intact state and after ovariectomy. Twelve female Fischer rats were randomized into ovariectomy and sham operation groups. A rat eater meter measured the feeding indexes for 15 days before and 25 days after ovariectomy, and later for 35 days with hormone therapy. We report: (a) mean body weight gain was linear before and up to ovariectomy, while exponential after ovariectomy; (b) increase in daily food consumption is mainly via an increase in food intake during the light phase; (c) light phase meal number remains unchanged, meal size significantly increases, with the resultant increase in overall food intake; (d) during the dark phase, meal size also significantly increases, but is accompanied by a proportional decrease in meal number, resulting in unchanged dark-phase food intake; and (e) estrogen restoration with either estradiol valerate or estradiol-progesterone combination, reversed the above changes. Data show that in the female Fischer 344 rat: (a) changes in daily rhythm in food intake are brought about by differential effects of the hormones on both meal size and meal number in both the total daily levels as well as in the dark-to-light distribution; (b) estadiol appears to have a tonic inhibitory effect on the light phase meal size and a phasic effect on the dark phase meal size and number, but no significant effect on the light-phase meal number; and (c) in the Fischer rats, progesterone augments estradiols effect on these indicies.

Ultra high performance liquid chromatography tandem mass spectrometric detection in clinical analysis of simvastatin and atorvastatin.

Simvastatin and atorvastatin belong to the group of hypolipidemic drugs, more exactly to the second generation of inhibitors of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They induce a significant reduction in total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides, therefore they are widely used in the treatment of hypercholesterolemia even of its severe form-familiar hypercholesterolemia. Simvastatin and atorvastatin as the most widely used statins in clinical treatment and their hydroxy-acid/lactone forms were determined by means of UPLC in connection with triple quadrupole mass spectrometer. Deuterium labeled reference standard compounds were used as internal standards for the quantitation. Separation was performed on Acquity BEH C18 (100 mm x 2.1 mm, 1.7 microm) using gradient elution by mobile phase containing acetonitrile and ammonium acetate pH 4.0, which is convenient in order to prevent interconversion of analytes. ESI in positive mode was used for the ionization of all compounds. Two SRM (selected reaction monitoring) transitions were carefully optimized for each analyte in order to get high sensitivity and selectivity. SPE on Discovery DSC-18 was used as a sample preparation step. Intra-day precision was generally within 10% RSD, while inter-day precision within 15% RSD. Method accuracy expressed as recovery ranged from 75 to 100%. The method was validated with the sensitivity reaching LOQ 0.08-5.46 nmol/l and LOD 0.01-1.80 nmol/l in biological samples. Atorvastatin, simvastatin, its metabolites and hydroxy-acid/lactone forms were monitored in human serum and in lipoprotein fractions (LDL, HDL and VLDL) at patients with end stage renal diseases.

Biochemical profile and survival in nonagenarians

OBJECTIVES Old age is associated with an increase in frequency of disorders involving virtually all organ systems, resulting in a rise of mortality. The aim of the project was to study the relationship between biochemical markers and all-cause mortality in a defined age group. DESIGN AND METHODS Thirty-eight nonagenarians, aged 92 +/- 2 (range 90-100) years entered the study. At the start of the study, a sample of peripheral blood and urine were obtained for analysis of 50 basic biochemical, hematologic and biologic parameters. The assessment was then repeated in 6 to 12 months intervals. The significance of difference between surviving subjects and those who died was examined by Mann-Whitney U test and the correlation between the variables was studied by Spearman rank correlation coefficient. RESULTS During the observation period, 21 of the studied subjects died leaving 17 persons still alive at the end of the study. The mean time from the first measurement to the death was 12 +/- 10 (range 0-33) months. The mean follow-up time in surviving subjects was 31 +/- 12 (range 4-45) months. Serum vitamin E and calcium were significantly higher, and serum alanine aminotransferase (ALT) and urinary neopterin were significantly lower in survivors compared to the subjects who died. No other parameters were significantly different in survivors and in persons who died. Urinary neopterin exhibited a significant negative correlation with serum sodium concentration (RS = -0.50, p < 0.01), but the other parameters did not correlate significantly. CONCLUSION In conclusion, among the parameters studied, differences between survivors and nonsurvivors were observed only for serum vitamin E, calcium, ALT and urinary neopterin. These findings may form a basis for prospective interventional trials in this group of patients.

Use of orchiectomy and testosterone replacement to explore meal number-to-meal size relationship in male rats

Because food intake is a function of meal number and meal size and because gender-related hormones are involved in feeding regulation, we explored effects of orchiectomy and testosterone replacement on the relationship between meal number and size and changes in resulting feeding patterns in adult male rats, randomized into orchiectomy and sham-operation groups. A rat eater meter measured feeding indexes for 1 wk before and 2 wk after castration and during 8 days of testosterone replacement. Orchiectomy leads to an immediate change in the meal number-to-size relationship, resulting in 1) change in pattern of feeding; 2) a significant decrease in dark-phase meal number; 3) a significant increase in dark-phase meal size, but insufficient to offset decrease in meal number, so total food intake significantly decreased during dark phase; 4) no significant change in light-phase meal number; and 5) an increase in meal size leading to an increased food intake during light phase, which offset decreased food intake in dark cycle and resulted in no net significant change in food intake after orchiectomy. Testosterone replacement acutely reversed effects of orchiectomy on meal number-to-meal size relationship, restoring feeding pattern. Data suggest that androgens immediately influence the meal number-to-meal size relationship. The speed of onset seen after orchiectomy suggests that the influence of testosterone on food intake may also occur partially via a nongenomic effect.Because food intake is a function of meal number and meal size and because gender-related hormones are involved in feeding regulation, we explored effects of orchiectomy and testosterone replacement on the relationship between meal number and size and changes in resulting feeding patterns in adult male rats, randomized into orchiectomy and sham-operation groups. A rat eater meter measured feeding indexes for 1 wk before and 2 wk after castration and during 8 days of testosterone replacement. Orchiectomy leads to an immediate change in the meal number-to-size relationship, resulting in 1) change in pattern of feeding; 2) a significant decrease in dark-phase meal number; 3) a significant increase in dark-phase meal size, but insufficient to offset decrease in meal number, so total food intake significantly decreased during dark phase; 4) no significant change in light-phase meal number; and 5) an increase in meal size leading to an increased food intake during light phase, which offset decreased food intake in dark cycle and resulted in no net significant change in food intake after orchiectomy. Testosterone replacement acutely reversed effects of orchiectomy on meal number-to-meal size relationship, restoring feeding pattern. Data suggest that androgens immediately influence the meal number-to-meal size relationship. The speed of onset seen after orchiectomy suggests that the influence of testosterone on food intake may also occur partially via a nongenomic effect.

Beneficial effect of plasma exchange in the treatment of toxic epidermal necrolysis: A series of four cases

Introduction: Toxic epidermal necrolysis (TEN) is a rare, life‐threatening disease with a high mortality rate that is linked to drug toxicity. There is a lack of data about the underlying pathophysiologic mechanisms and treatment options. The only widely accepted treatment of TEN is withdrawal of the offending drug followed by supportive care. The potential roles of corticosteroids, intravenous immunoglobulin (IVIG) and plasmapheresis (TPE) remain controversial. Aims: We present four patients with severe TEN (all with >80% involvement of body surface) who were treated with TPE following unsuccessful treatment with corticosteroids/IVIG. Methods: TPE was performed using a COBE Spectra blood cell separator. ACD‐A was used as anticoagulant fluid and the target‐washed plasma volume was one body volume. Plasma was replaced by a 5% solution of human albumin + Ringers lactate. Results: The mean number of TPE sessions was 5.25 ± 2.22 (range 3–8). Drugs were implicated as an etiologic agent in each case. TPE led to prompt improvement of acute condition and general health as well as halting of disease progression. Additionally, the restoration of the epithelium began in all four patients. Conclusion: Plasmapheresis should be considered as an alternative treatment modality for patients with the most severe form of TEN if initial treatment with other agents, including corticosteroids and/or IVIG, fails. Drugs were suspected to be the cause of TEN in all four cases. J. Clin. Apheresis, 2012.

High-dose chemotherapy followed by autologous stem cell transplantation changes prognosis of IgD multiple myeloma

Immunoglobulin D (IgD) multiple myeloma (MM) is a rare plasma cell disorder constituting less than 2% of all MM cases. Survival of patients with IgD MM is generally shorter than that of patients with other types of monoclonal (M-) protein. We have retrospectively analyzed patients with IgD MM participating in clinical trials of the Czech Myeloma Group. Twenty-six IgD MM patients treated between 1996 and 2006 were identified, 14 (54%) men and 12 (46%) women. The median age was 61 years (range: 37–79 years). Ten of 26 patients (39%) were treated with first-line high-dose chemotherapy (HDCT) using melphalan 200 mg/m2 followed by autologous stem cell transplantation (ASCT). Thirteen of 26 patients (50%) received conventional chemotherapy (CHT), mostly melphalan and prednisone or a vincristine/doxorubicin/dexamethasone (VAD) regimen. Treatment responses were evaluable for 23 of 26 (89%) patients. All HDCT patients had treatment responses, including seven patients (70%) with complete responses and three patients (30%) with partial responses. The median progression-free survival was 18 months for HDCT patients and 20 months for CHT patients. The median overall survival (OS) for all patients was 34 months. The median OS for the HDCT group has not yet been reached (70% of the patients are still alive). In contrast, the median OS for CHT patients was only 16 months. The difference in OS between the two groups was statistically significant (P=0.005). In conclusion, the overall response rate for patients with IgD MM aged 65 years or less treated with HDCT and ASCT is similar to that seen in other MM types.

Plasma albumin levels correlate with decreased microcirculation and the development of skin defects in hemodialyzed patients

OBJECTIVES Difficulty healing wounds and skin defects is a frequent problem in patients on chronic hemodialysis (HD) because of malnutrition, inflammation, and atherosclerosis (MIA) syndrome. The aim of the present study was to estimate the influence of peripheral blood flow changes during HD on the development of foot defects and its relationship to plasma albumin levels. METHODS Peripheral skin blood flow was measured using a laser Doppler line scanner in 10 different areas of the dorsal part of the instep and the toes of each foot before and during HD with ultrafiltration (897 +/- 465 mL/procedure) in 31 HD patients (10 female, 21 male; age 36-79 y, body mass index = 28 +/- 5.0). No skin defects or apparent acute disease or infection were detected in any patient at the time of laser Doppler line scanner measurement. The feet of the patients were clinically re-examined carefully over the next 18 mo. RESULTS We found a significant and constant decrease of skin blood flow during the HD procedure (P < 0.001). Skin blood flow was significantly correlated with serum albumin level both before HD (r = 0.36, P = 0.05) and during HD (r = 0.47, P = 0.007). Skin defects developed in 11 patients, with significantly lower skin blood flow during the 18-mo follow-up period. A significantly larger number of patients who had normal perfusion remained defect-free in comparison to patients with critical perfusion (93% versus 38%, P = 0.002, Kaplan-Meier analysis). CONCLUSION Skin blood flow may be impaired in HD patients. The apparent malnutrition and inflammation in HD patients are likely responsible for the decreased skin blood flow and the development of the difficulty to heal skin defects and wounds.

Safety and Tolerability of Long Lasting LDL-apheresis in Familial Hyperlipoproteinemia

Abstract:  The aim of this work is to arbitrate the incidence of side effects and tolerability of long lasting LDL‐apheresis in familial hyperlipoproteinemia. 1200 procedures were performed and the last 463 of them were evaluated. An immunoadsorption method of LDL‐apheresis was used (continuous blood cell separator Cobe Spectra; secondary device: automated adsorption‐desorption ADA, Medicap; absorption columns: Lipopak). As a whole, 6.26% adverse events were found and subsequently resolved by standard symptomatic therapy. Vaso‐vagal reactions (symptoms of neurovegetative lability) were the most common adverse effects, presented as malaise, weakness, slight and short‐term drop in blood pressure or other general signs. They were all well controlled by symptomatic therapy. We conclude that LDL‐apheresis in the hands of experienced personnel is a safe procedure. An acceptable procedure duration limit, balancing the possibility to achieve a targeted cholesterol level while still maintaining an acceptable patient tolerance, was confirmed to be 4 hours.

Cholesterol metabolism in active Crohn's disease

ZusammenfassungErniedrigte Cholesterin-Spiegel gelten als typisches Merkmal einer schweren Erkrankung und sind mit einer schlechten Prognose vergesellschaftet. Der Morbus Crohn ist eine entzündliche Erkrankung, die mit verschiedenen Störungen des Stoffwechsels einhergeht. In den vergangenen Dekaden haben verschiedene klinische Studien einen Zusammenhang zwischen dem Lipidstoffwechsel und der systemischen Entzündung etabliert. In unserer Studie haben wir nicht nur das Cholesterin-Profil (Cholesterin, LDL und HDL Cholesterin) im Plasma bestimmt, sondern auch Veränderungen im Cholesterin-Absorptions- beziehungsweise -Synthese-Prozess durch Bestimmung der Konzentrationen der Nicht-Cholesterin-Sterole im Serum untersucht. Die Plasmakonzentrationen des Gesamtcholesterins, des LDL- und HDL-Cholesterins sowie der Nicht-Cholesterin-Sterole (Squalene, Lathosterol, Campesterol, Sitosterol) wurden bei 24 Patienten mit aktivem Morbus Crohn in einer Zeitspanne von 28 Tagen erhoben. Die Plasmakonzentrationen des Gesamtcholesterins (p < 0,001) und des LDL-, und HDL-Cholesterins (p < 0,05) waren signifikant niedriger als die bei Kontrollpersonen erhobenen Werte. Im Vergleich zur Kontrolle hatten die Patienten mit aktivem Morbus Crohn signifikant niedrigere Plasmaspiegel von Lathosterol (p < 0,001) mit gleichzeitig nicht signifikant erhöhten Konzentrationen von Squalene. Die Campesterol-Plasmakonzentrationen waren signifikant (p < 0,001) erniedrigt. Sitosterol auch – aber nicht signifikant. Die aktive Phase des Morbus Crohn ist durch Veränderungen des Lipidstoffwechsels – vor allem von Cholesterin – gekennzeichnet. Unsere Ergebnisse zeigen Plasmakonzentrationen der Nicht-Cholesterin-Sterole außerhalb der Norm und sprechen dafür, dass die Cholesterin-Synthese und -Absorption beim aktiven Morbus Crohn verändert ist.SummaryHypocholesterolemia has been investigated as a typical feature of critical illness and is connected with poor prognosis. Crohns disease is an inflammatory process and is associated with several metabolic disturbances. In recent decades clinical studies have established a link between lipid metabolism and systemic inflammation. In our study we examined the serum profile of cholesterol (total cholesterol, LDL- and HDL-cholesterol) and changes in the cholesterol absorption/synthesis process by determination of plasma non-cholesterol sterol (squalene, lathosterol, campesterol, sitosterol) concentrations. Serum concentrations of total cholesterol, LDL- and HDL-cholesterol and non-cholesterol sterols were evaluated in 24 patients with active Crohns disease during a period of 28 days. We detected lower serum levels of total cholesterol (P < 0.001), LDL- and HDL-cholesterol (P < 0.05) in the patients with active Crohns disease than in the control group. In addition, the patients had significantly lower plasma levels of lathosterol (P < 0.001) and higher concentrations of squalene, although without significant differences. A significant decrease of campesterol plasma levels (P < 0.001) was detected, but lower plasma concentrations of sitosterol were without statistical significance. The active phase of Crohns disease is characterized by altered metabolism of lipids, mainly of cholesterol. Our results show abnormalities in plasma concentrations of non-cholesterol sterols and provide evidence that the process of cholesterol synthesis and absorption is altered in active Crohns disease.