Membrane permeability related physicochemical properties of a novel gamma-secretase inhibitor

Pharmaceutical profiling studies were conducted on a novel prototype γ-secretase inhibitor, to determine the potential of its oral absorption. Such compounds can be of use in the treatment of Alzheimer’s disease (AD). The studies included determination of solubility, dissociation constant (pK_a), octanol/water partition coefficient (log P) and the capacity factor (k_IAM′) on immobilized artificial membrane (IAM) chromatographic columns. The compound is very slightly solubility in water (120 ± 50 μg/mL) but the solubility increased considerably in basic medium (270 ± 60 μg/mL). The compound exhibited pK_a of (10.36 ± 0.11); and log P of (3.36 ± 0.16) determined by shake-flask method and (3.31 ± 0.01) determined by high performance liquid chromatography (HPLC). The experimentally determined log P values correlated well with the calculated one of 3.44. The observed log k_IAM′ value of (2.79 ± 0.04) indicates that the compound can reasonably be expected to have high membrane permeability, and therefore, good absorption profile if taken orally. This conclusion is also supported by other parameters determined.     

Hydrophobicity of β-adrenoceptor blocking agents: Study of correlations between retention in reversed-phase HPLC systems and octanol-water partition constants

The capacity factors (k′) of seven β-adrenoceptor blocking agents in six different reversed-phase HPLC systems have been determined. Octanol-aqueous buffer (pH 7.4) partition constants (P) for these blocking agents were also obtained. By using target factor analysis (TFA), good empirical correlations between the log k′ and log P were derived. The resulting hydrophobicity order agrees well with the metabolic elimination pathways of these drugs.     

Relationship between polysorbate 80 solubilization descriptors and octanol-water partition coefficients of drugs

The molar solubilization capacities (κ) and the molar micelle–water partition coefficients (K_M^N) in Polysorbate 80 of several drugs (including barbiturates, steroids, and benzoic acid derivatives) are related to their log octanol–water partition coefficients (log P). Both κ and K_M^N values were calculated from solubility versus Polysorbate 80 concentration profiles, which were either experimentally determined or obtained from the literature. There is a linear relationship between log P of the tested compounds and the logarithm of the molar micelle–water partition coefficient (log K_M^N). On the other hand molar solubilization capacities are nearly independent of log P. It is shown that the ability of Polysorbate 80 to solubilize a drug can be predicted from its log P value.     

Chronic administration of losartan plus hydrochlorothiazide improves vascular status in young cardiomyopathic hamsters

The combination of an angiotensin II receptor antagonist and a thiazide has been used extensively in the treatment of patients with overt heart failure. The effect of this combination on the vascular wall early in the disease, however, has not been investigated. To evaluate this effect, the vascular status of 3-month-old cardiomyopathic hamsters was assessed after daily administration of a combination of losartan (25 mg/kg, p.o.) and hydrochlorothiazide (6.5 mg/kg, p.o.) over an 8-week period. Age-matched golden hamsters were used as healthy controls. The contractile response of aortic rings to endothelin-1 was significantly higher in cardiomyopathic hamsters than in control animals. Concentration–response curves for the endothelin-1-induced contraction were displaced to the right after hydrochlorothiazide+losartan treatment (toward the curves for healthy controls); however, E_max from treated hamsters was significantly reduced when compared to E_max from untreated cardiomyopathic animals (1.016±0.073 vs. 1.346±0.153 g, P<0.05, n=6). No significant differences in the EC₅₀ values from these curves were observed between hydrochlorothiazide+losartan treated and untreated cardiomyopathic animals (2.90±0.95 vs. 1.10±0.85 nM, P>0.05). The acetylcholine-induced relaxation observed in cardiomyopathic animals was not improved after treatment with hydrochlorothiazide+losartan or hydrochlorothiazide alone, but the combination of these drugs increased significantly the basal production of nitric oxide (NO). Angiotensin-converting enzyme activity increased in plasma (from 29.9±1.23 to 41.16±1.82 nmol mg⁻¹ min⁻¹, n=8, P<0.05) but decreased in the aorta (from 0.33±0.02 to 0.25±0.017 nmol mg⁻¹ min⁻¹, n=6, P<0.05) after treatment with hydrochlorothiazide+losartan. In addition, the combination of these drugs reduced the heart-to-body mass ratio (3.96±0.07 for treated vs. 5.01±0.20 mg/g for untreated animals, n=7, P<0.05), and the thickness of the aortic media (0.076±0.003 for treated vs. 0.149±0.009 mm for untreated animals, n=8, P<0.05). Although hydrochlorothiazide alone lowered systolic blood pressure to the same level achieved with both drugs in combination (from 166±10 for untreated cardiomyopathic animals to 84±1 mm Hg for hydrochlorothiazide+losartan, and 80±5 mm Hg for hydrochlorothiazide alone, P<0.05), no significant reduction in heart-to-body mass ratio was observed in animals treated with the diuretic alone (P>0.05). In conclusion, in this model of heart failure, chronic hydrochlorothiazide+losartan administration normalizes the vascular responses to endothelin-1, improves basal vascular tone, and prevents the development of cardiac and vascular hypertrophy.     

Inhibition of 1,2-dimethylhydrazine-induced oxidative DNA damage in rat colon mucosa by black tea complex polyphenols

The effect of black tea polyphenols on 1,2-dimethylhydrazine (DMH)-induced oxidative DNA damage in rat colon mucosa has been investigated. Fischer 344 rats were treated orally with thearubigin (TR) or theafulvin (TFu) for 10 days (40 mg/kg), injected ip with DMH (20 mg/kg) or saline and sacrificed 24 hr after DMH administration. The levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured in colonic mucosa DNA and expressed as a ratio relative to 2′-deoxyguanosine (2dG). Control rat mucosa had 8-OHdG values of 1.12 ± 0.14/10⁵ dG (mean ± SEM, N=11), whereas DMH-treated rats significantly higher values (1.52 ± 0.14/10⁵ dG, N=26, P<0.05). Pretreatment of rats with TR had significantly inhibited DMH-induced oxidative DNA damage 0.99 ± 0.09/10⁵ dG, N=10, P<0.05) and a similar, although less marked, effect was observed with TFu (1.15 ± 0.19/10⁵, N=9, P=0.06). These findings confirm that DMH causes oxidative DNA damage in the colon mucosa of rats and demonstrate that this effect is prevented by the consumption of complex polyphenols from black tea.     

Potent antagonism of 5-HT(3) and 5-HT(6) receptors by olanzapine.

The interaction of the psychotropic agent olanzapine with serotonin 5-HT₃ and 5-HT₆ receptors was investigated. Olanzapine did not contract the isolated guinea pig ileum, but blocked contractions induced by the 5-HT₃ receptor agonist 2-methyl serotonin (2-CH₃ 5-HT) with a pK_B value of 6.38±0.03, close to the affinity of the 5-HT₃ receptor antagonist ondansetron. The atypical antipsychotic risperidone (1 μM) did not significantly inhibit 2-CH₃ 5-HT-induced contractions. Olanzapine had high affinity (pK_i=8.30±0.06) for human 5-HT₆ receptors in radioligand binding studies. Olanzapine did not stimulate [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding to the G protein G_s in cells containing human 5-HT₆ receptors, but inhibited 5-HT-stimulated [³⁵S]GTPγS binding (pK_B=7.38±0.16). Among other antipsychotics investigated, clozapine antagonized 5-HT₆ receptors with a pK_B=7.42±0.15, ziprasidone was three-fold less potent, and risperidone, quetiapine and haloperidol were weak antagonists. Thus, olanzapine was not an agonist, but was a potent antagonist at 5-HT₆ receptors and had marked antagonism at 5-HT₃ receptors.     

Effects of Fe on ion channels: Na channel, delayed rectified and transient outward K channels

The effects of Fe²⁺ on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7–14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe²⁺, the activation voltage threshold of transient outward K⁺ currents (I_A) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V_1/2 was 2.44±1.14 mV (n=15) in control, whereas 1.79±1.53 (n=15), −2.96±0.92 (n=14), −5.11±1.31 (n=13), −9.05±1.64 mV (n=12) in 1, 10, 100 and 1000 μ Fe²⁺, respectively. Differences between two groups were significant (P<0.05, n=12–15), except for that between the control and 1 μ (P>0.05, n=15). (2) Fe²⁺ caused a left shift of the current–voltage curves of steady-state inactivation of I_A in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10–13). The V_1/2 were −70.71±1.23 (n=13), −71.14±1.37 (n=13), −78.21±1.17 (n=11), −84.61±1.34 (n=12), and −89.68±2.59 mV (n=10) in control, 1, 10, 100 and 1000 μ Fe²⁺, respectively. Fe²⁺ also shifted the current–voltage curves of Na⁺ channel steady-state inactivation to more negative depolarization potentials in parallel, with V_1/2, −67.37±1.33 mV (n=12) in control, and −67.52±1.28 mV (n=12), −68.24±1.61 mV (n=10), −71.58±1.45 mV (n=10), −76.65±1.76 mV (n=9) in 1, 10, 100 and 1000 μ Fe²⁺ solutions, respectively. (3) In Fe²⁺ solutions, the recovery from inactivation of I_A was slowed. (4) With application of different concentrations of Fe²⁺, the voltage threshold of activation of delayed rectified outward K⁺ currents (I_K) was decreased, while Fe²⁺ showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe²⁺ is a dose- and voltage-dependent, reversible modulator of I_A, I_K and Na⁺ channels. The results will be helpful to explain the mechanism of Fe²⁺ physiological function and Fe²⁺ intoxication in the central nervous system.     

Chromatographic retention of drug molecules on immobilised liposomes prepared from egg phospholipids and from chemically pure phospholipids

The partitioning of a chemically diverse set of drugs into liposomes was studied by immobilised liposome chromatography (ILC). For this purpose liposomes composed of (i) purified egg phospholipids (EPL), (ii) synthetic phosphatidylcholine (PC), (iii) PC–synthetic phosphatidylethanolamine (PE) 80:20 (mol/mol) and (iv) PC–synthetic phosphatidylserine (PS) 80:20 (mol/mol) were immobilised in gel beads by freeze–thawing. The drug partitioning was assessed from the retention volume, which was expressed as a capacity factor, K_s, normalised with respect to the amount of immobilised phospholipid. The drug retention on EPL, PC and PC–PE liposomes was very similar, whereas the negatively charged PC–PS liposomes increased the retention of positively charged and decreased retention of negatively charged drugs. The partitioning of drugs on liposome columns (log K_s) versus their octanol–water partitioning (log P_oct) showed three separate rectilinear relationships, depending on the charge of the compound (neutral, positive, or negative). Statistical analysis (ANCOVA) proved that the lines had similar slopes. Repeated analysis of four reference compounds showed a low variation (<0.12 log units) over time (about 250 days). A close relationship was observed between the drug retention in short EPL columns with a low content of phospholipids and the retention in longer standard EPL columns. The short ‘quick screen bilayer columns’ permit analysis of highly lipophilic compounds within 30 min and are thus applicable for medium-throughput screening in drug discovery settings. A very strong rectilinear relationship (r²=0.95, n=13) between log K_s (EPL) and published liposome partitioning data (log D_mem) confirmed that the ILC drug retention reflects the drug partitioning into the lipid bilayers. A moderate to fair rectilinear relationship was observed between the normalised retention on PC, PC–PE and EPL liposomes (r²=0.79, 0.86 and 0.85, respectively, n=24) and corresponding published log k′_IAM data obtained on immobilised artificial membrane (IAM) columns. Transport across Caco-2 cell monolayers (log P_c) showed curvilinear relationships with log K_s, log k′_IAM, log P_oct and log D_oct. The drug fraction absorbed in humans showed a similar relationship to log K_s values as to surface plasmon resonance signals representing drug–liposome interaction (Danelian et al., 2000 J Med Chem, 43, 2083–2086).     

Reversed-phase high-performance liquid chromatography for evaluating the distribution of pharmaceutical substances in suppository base-phosphate buffer system

The aim of this study was to assess the potency of the reversed-phase high-performance liquid chromatography (RP-HPLC) for in vitro evaluation of the distribution behavior of common drugs between one of the generally used suppository bases Witepsol H₁₅ and the rectal liquid which is imitated by a phosphate buffer, pH 7.2. The distribution coefficients (log K) of nine compounds — paracetamol, caffeine, diclofenac, propyphenazone, indomethacin, codeine base, codeine phosphate, phenobarbital acid and phenobarbital sodium salt were determined by the classical ‘shake-flask’ method followed by RP-HPLC quantitative assay. The capacity factors log k′ of the compounds were determined on reversed-phase C₁₈ column at a number of methanol–5 mM phosphate buffer, pH 7.2 mobile phases containing different percentages of methanol (φ_MeOH). The apparent capacity factors log k′_w^app were derived by extrapolation of the methanol concentration to zero and using the correction for ionization, the real capacity factors log k′_w were calculated. The lipophilicity of the compounds was assessed by the partition coefficients CLOGP and the distribution coefficients CLOGD_7.2, calculated for the n-octanol–water system. Correlations between log k′_w and CLOGP, log k′_w^app and CLOGD_7.2, log k′_w^app and log K were found. The last correlation indicated that the parameter log k′_w^app was suitable for evaluating the distribution behavior of the studied drugs in the examined Witepsol H₁₅-rectal liquid system. The predictive power of this correlation was tested by a set of nine non-congeners. It was shown that the classical ‘shake-flask’ method for determination of the distribution behavior of the studied drugs between the suppository base Witepsol H₁₅ and the phosphate buffer, pH 7.2 might be replaced by the RP-HPLC technique due to its priorities of rapid, stable and reproducible experiments.     

Evaluation of the effect of organic modifier on pK values of diuretics in mobile phases used in LC

The dissociation pK values of a series of diuretics in 10, 30, 40, 50 and 70% (w/w) acetonitrile–water mixed solvents at 25°C were determined according to the criteria endorsed by IUPAC. The series of diuretics chosen includes compounds with differences in molecular structures and physico-chemical properties. Acidic compounds (loop diuretics, such as furosemide, bumetanide and ethacrynic acid), weakly acidic (thiazides, such as chlorthiazide and trichlormethiazide), neutral (aldosterone antagonists, such as canrenone) and basic compounds (potassium-sparing diuretics, such as amiloride and triamterene) were all considered. The variation of the pK values obtained over the whole composition range studied can be explained by taking into account the preferential solvation of ionizable compounds in acetonitrile–water mixtures. Moreover, in order to obtain pK values in any of the unlimited number of possible binary solvent acetonitrile–water mixtures, relationships between pK values and different bulk properties were examined, and the linear solvation energy relationships method (LSER) was applied to study the correlations of pK values with the solvatochromic parameters π*, and β of acetonitrile–water mixtures. The equations obtained allowed calculation of the pK values of diuretics in any acetonitrile–water mixtures up to 70% (w/w) and thus permitted the acid–base behaviour of these important substances in the widely used acetonitrile–water media to be known.     

Determination of the ionization constants of 4-iodo-2,6-dimethylphenylcarbamoylmethyl iminodiacetic acid

In order to develop a radiopharmaceutical for hepatobiliary scintigraphy with better hepatobiliary properties new ligand for complexation of ^99mTc, 4-iodo-2,6-dimethylphenylcarbamoylmethyl iminodiacetic acid (METHYLIODIDA), was synthesized. Acid–base equilibria of METHYLIODIDA were studied potentiometrically, because these data are important for determination of complex formation conditions. It was established that METHYLIODIDA undergoes a complex acid–base equilibrium due to its zwitterionic nature and four proton-binding sites. The stoichiometric ionization constants were determined at 25 °C and constant ionic strength 0.1 M (NaClO₄): pK₁=1.7±0.1; pK₂=2.44±0.07; pK₃=6.29±0.02 and pK₄=10.91±0.06, respectively.     

Relation between retention factors of immunosuppressive drugs in microemulsion electrokinetic chromatography with biosurfactants and octanol-water partition coefficients

Retention (capacity) factors (k′ values) of immunosuppressive drugs were determined in microemulsion electrokinetic chromatography (MEEKC) systems as a tool for the indirect estimation of partition coefficients (P_OW) between 1-octanol and water. The microemulsions were based on phosphatidylcholine (PC) and bile acids (BAs) as biosurfactants and isopropyl myristate (IPM) as oil. Immunosuppressants were azathioprine (AZA), mycophenolate mofetil (MMF), tacrolimus (FK506) and cyclosporine A (CyA). Capacity factors of the analytes were determined from electrophoretic mobilities using an aqueous phosphate buffer (20 mM; pH 7.5) for all the systems. Retention was compared with that in the most commonly used microemulsion based on sodium dodecyl sulphate (SDS). log P_OW versus log k′ calibration lines were constructed using reference compounds with known P_OW. In addition, data of log P_OW of the immunosuppressants were determined by partitioning between octanol and water, and were calculated by the aid of computer program. A different sequence of log P_OW for two analytes was found in the biosurfactant-based systems compared with the SDS-containing one. Excellent agreement was observed between the log P_OW values derived from the microemulsions containing deoxycholate compared with the data determined by partitioning between octanol and water. It was concluded that the retention factors in the systems with biosurfactants are good estimators for the partitioning in biological systems.     

某三甲精神病院利培酮血药浓度监测现状及影响因素分析

目的：通过分析某三甲精神病院利培酮（risperidone,RIS）血药浓度监测（therapeutic drug monitoring,TDM）现状,探索RIS血药浓度影响因素。方法：采用回顾性分析方法,收集2019年7月至2020年7月进行RIS TDM住院患者的检测结果及相关病例资料,分析利培酮、活性代谢物9-羟基利培酮（9-OH-RIS）以及总活性成分（active moiety,AM）血药浓度值的分布情况,探索日剂量、性别、年龄对血药浓度的影响以及体质量和体质指数（body mass index,BMI）与血药浓度的相关性。结果：1 007例患者共行RIS TDM 4 458例次,平均监测次数为4.47次,29.99%例次监测结果不在建议的参考范围（20~60 ng·mL^-1）内,临床实际RIS血药浓度值（C_RIS）、9-OH-RIS血药浓度值（C_9-OH-RIS）以及AM血药浓度值（C_AM）的参考值范围分别为（0.45~39.79）、（8.43~74.87）和（11.74~97.23） ng·mL^-1。C_AM与日剂量具有相关性（ρ=0.536,P<0.01）,C_9-OH-RIS与日剂量具有相关性（ρ=0.570,P<0.01）,C_RIS与日剂量呈较弱的相关性（ρ=0.192,P<0.01）。女性较男性有更高的C_AM与AM浓度剂量比（C/D）值,不同年龄段患者C_AM与C/D值存在差异。体质量与C_AM（ρ=-0.065,P<0.01）和C/D值（ρ=-0.098,P<0.01）呈负相关,与RIS/9-OH-RIS比值（ρ=0.037,P=0.014）呈正相关。BMI与C_AM（ρ=-0.054,P<0.01）和C/D值（ρ=-0.033,P=0.026）呈负相关。结论：临床实际C_AM参考值范围相比于AGNP-TDM专家组推荐的参考范围更宽;日剂量、性别、年龄、体质量和BMI是影响C_AM以及C/D值的重要因素,这些因素或可以为RIS临床个体化用药提供参考。     

Purification and biological effects of C-type lectin isolated from Bothrops insularis venom

Bothrops insularis is a snake from Queimada Grande Island, which is an island located about 20 miles away from the southeastern coast of Brazil. Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. Its C-type lectin is involved in several biological processes including anticoagulant and platelet-modulating activities. We purified the C-type lectin (BiLec) from Bothrops insularis venom and investigated its effect in the isolated kidney. BiLec was purified after two chromatographic steps; firstly, the whole venom was submitted to an HPLC molecular exclusion chromatography followed by a second purification through affinity chromatography. B. insularis lectin (BiLec) was studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. The concentration of 10 μg/mL increased perfusion pressure (PP; control₆₀=108.27±4.9; BiLec₆₀=112.9±5.4 mmHg; *p<0.05) and renal vascular resistance (RVR; control₆₀=5.38±0.51; BiLec₆₀=6.01±0.57 mmHg; *p<0.05). The urinary flow reduced significantly at 90 and 120 min of perfusion (UF; control₁₂₀=0.160±0.020; BiLec₁₂₀=0.082±0.008 mL g⁻¹ min⁻¹; *p<0.05). Glomerular filtration rate (GFR; control₁₂₀=0.697±0.084; BiLec₁₂₀=0.394±0.063 mL g⁻¹ min⁻¹; *p<0.05) diminished only at 120 min. BiLec did not change the percentage of sodium (TNa⁺), potassium (TK⁺) and chloride tubular transport (TCl⁻). The histological alterations probably reflected direct injury on glomerular and tubular renal cells, as demonstrated by the rise in permeability of glomerular endothelial cells, revealed by the presence of a proteinaceous material in the Bowman space. We postulate that the C-type lectin B. insularis promoted its effects probably through interactions with endothelial cells or through the release of other mediators by tubular, mesangial and endothelial cells.     

An observation on the mercury contents of scalp hair in the urban residents of South Korea

The average value of total mercury (THg) in scalp hair of male residents in Seoul city was 1.7±0.18 ppm (mean±S.E.) and that of methylmercury (MeHg), 1.0±0.12 ppm (58.8% THg). In female, level of THg was 1.1±0.15 ppm and MeHg was 0.5±0.14 ppm (45.5%). Mercury was found more in the scalp hair of male than female (P<0.01). THg/MeHg increased with age of subjects in male (P<0.01), but not female. Coefficients of correlation (r) between THg and MeHg contents in scalp hair of male was +0.877 (P<0.01) and that of female was +0.508 (P<0.01), respectively.     

Differential effects of sulindac on renal hemodynamics and function in the rat

Renal hemodynamics were studied using an electromagnetic perivascular flow sensor in anesthetized rats injected i.v. with vehicle, 5 or 10 mg/kg body weight (b.w.) sulindac. No hemodynamic changes occurred with vehicle (n=6), but mean arterial pressure was significantly decreased (by 15 mmHg) with sulindac (n=12). In the 5 mg/kg b.w. sulindac group (n=7), renal blood flow progressively and significantly increased from 7.88±0.36 to 8.98±0.58 ml/min, except during concomitant intrarenal infusion of 3 mg/kg b.w. per h proadifen (n=7). The pressure limits for efficient and no renal blood flow autoregulation remained unchanged (approx. 100 and 80 mmHg, respectively). In the 10 mg/kg b.w. sulindac group (n=5), renal blood flow did not change but autoregulatory pressure limits were lowered by 10 mmHg 2 h after treatment (P<0.025). Also, Na⁺ retention was marked. Prostanoid excretion in urine was significantly reduced with either dose but basal plasma renin activity was not (about 8 ng/ml per h; n=15). When plasma renin activity was enhanced after a reduction in renal perfusion pressure (n=21), it was decreased from 11.5±1.2 to 7.4±0.2 ng/ml per h only by 10 mg/kg b.w. sulindac (P<0.05; n=6). In conclusion, differential effects of sulindac on renal hemodynamics, Na⁺ excretion and plasma renin activity were demonstrated. Renal hemodynamic changes could be related in part to the cytochrome P-450 arachidonic acid pathway.     

Comparative toxicity of sulfur mustard and nitrogen mustard on tracheal epithelial cells in primary culture

Sulfur mustard (SM) and mechlorethamine (HN2) are two alkylating agents. SM represents a potential chemical warfare agent and HN2 is used in cancer chemotherapy. Based on the similarities of their action, although few comparative studies of their effects have been performed on the same model, many compounds effective against HN2 side-effects have been proposed, unsuccessfully, against SM-induced lesions. We performed this study to compare the toxic effects of these two alkylating agents on rabbit tracheal epithelium in primary culture. Using neutral red uptake, we evidenced that for a time of contact of 24 hr, HN2 LC₅₀ was significantly lower than SM LC₅₀ (0.034±0.009 and 0.132±0.023 m , respectively; P<0.001). On the other hand, for exposure at 10⁻³ , the time necessary to decrease the cell viability rate to 50% was shorter with SM than with HN2 (11±1 min and 54±2 min, respectively; P<0.0001). These two alkylating agents induced apoptosis which was evidenced by DNA ladder and by 4′,6-diamidino-2-phenylindole (DAPI) DAPI staining. The apoptosis rates were time and dose dependent for the two toxics: mild doses induced apoptosis, while higher doses induced necrosis.     

Bioanalysis of age-related changes of lipid metabolism in nonagenarians

The aim of the present study was the bioanalysis of lipid metabolism in the aged patients and to study the relationship between these biochemical markers and longevity. Eleven nonagenarians, nine women and two men, aged 94±3 years and ten control patients, six women and four men, aged 84±5 years, followed at the Department of Metabolic Care and Gerontology, Charles University, Teaching Hospital entered the study. All subjects were self-sufficient, without major illnesses and free living. At the start of the project the free fatty acids (FFA), thiobarbituric reactive substances (TBARS), retinol, alpha tocopherol, ascorbic acid, cholesterol, triacylglycerols, phospholipids in serum, in lipoprotein fractions and fatty acids (FA) and phospholipids in erythrocyte membrane were determined. We used capillary gas chromatography for determination of fatty acids. Retinol and alpha tocopherol were analysed by reversed-phase high-performance liquid chromatography, other parameters were determinated spectrophotometrically or spectrofluorometrically. We found significantly higher LDL polyunsaturated fatty acids (PUFA) 22:4n−3 (P=0.028) and 22:6n−3 (P=0.018) and a significant increase of HDL alpha tocopherol/cholesterol ratio (P=0.034) in nonagenarians. There were not any significant differences in erythrocyte membrane fatty acids and phospholipids. In serum we found significantly higher level of TBARS (3.22±1.22 vs 1.98±0.71 μmol/l, P=0.012) in nonagenarians, other parameters were not changed significantly. The higher concentration of PUFAs in LDL and alpha tocopherol in HDL might be parameters related to longevity.