Is There a Link Between Changes in Levels of Hepcidin and Stroke?

BACKGROUND: Iron is an essential element for the living body. It is well known that iron homeostasis disorders are important in two ways--its deficiency and its overload lead to several pathologies. METHODS: We measured 17 patients with iron deficiency anemia (IDA); 19 with anemia of chronic diseases (ACD); 15 with ischemic stroke (IS). The results were compared to a previously selected control group. For evaluation of iron metabolism status, we measured serum iron levels, ferritin, and soluble transferrin receptors. For inflammation, serum interleukin-6 and hsCRP were measured. Serum hepcidin quantification was performed using a previously validated immunosorbent method. Ferritin was measured by an ECLIA method; serum iron on AAS; hsCRP using a nephelometric analysis. RESULTS: We found statistically significant elevated serum hepcidin levels in patients with ACD and IS compared to the control group (p < 0.001). Patients with IDA had statistically significant lower hepcidin levels compared to the control group (p < 0.001). Serum ferritin levels in the IS group was higher compared to the control and other groups (p < 0.001). The lowest ferritin concentrations were established in the IDA group compared to the control (p < 0.001). We found a strong correlation between serum hepcidin and ferritin levels in the IS group (r = 0.583; p < 0.001). CONCLUSIONS: Quantification of serum hepcidin levels might be used as a link for prediction of acute ischemic stroke and future therapeutic influences.

Microbiota and mitobiota. Putting an equal sign between mitochondria and bacteria.

The recent revival of old theories and setting them on modern scientific rails to a large extent are also relevant to mitochondrial science. Given the widespread belief that mitochondria are symbionts of ancient bacterial origin, the processes inherent to mitochondrial physiology can be revised based on their comparative analysis with possible involvement of bacteria. Such comparison combined with discussion of the role of microbiota in pathogenesis allows discussion of the role of "mitobiota" (we introduce this term) as the combination of different phenotypic manifestations of mitochondria in the organism reflecting pathological changes in the mitochondrial genome. When putting an equal sign between mitochondria and bacteria, we find similarity between the mitochondrial and bacterial theories of cancer. The presence of the term "bacterial infection" suggests "mitochondrial infection", and mitochondrial (oxidative) theory of aging can in some way be transformed into a "bacterial theory of aging". The possible existence of such processes and the data confirming their presence are discussed in this review. If such a comparison has the right to exist, the homeostasis of "mitobiota" is of not lesser physiological importance than homeostasis of microbiota, which has been so intensively discussed recently.

Whole-genome-wide association study in the Bulgarian population reveals HHAT as schizophrenia susceptibility gene.

OBJECTIVE: Schizophrenia, the most common major psychiatric disorder (or group of disorders), entails severe decline of higher functions, principally with alterations in cognitive functioning and reality perception. Both genetic and environmental factors are involved in its pathogenesis; however, its genetic background still needs to be clarified. The objective of the study was to reveal genetic markers associated with schizophrenia in the Bulgarian population. METHODS: We have conducted a genome-wide association study using 554 496 single nucleotide polymorphisms (SNPs) in 188 affected and 376 unaffected Bulgarian individuals. Subsequently, the 100 candidate SNPs that revealed the smallest P-values were further evaluated in an additional set of 99 case and 328 control samples. RESULTS: We found a significant association between schizophrenia and the intronic SNP rs7527939 in the HHAT gene (P-value of 6.49×10 with an odds ratio of 2.63, 95% confidence interval of 1.89-3.66). We also genotyped additional SNPs within a 58-kb linkage disequilibrium block surrounding the landmark SNP. CONCLUSION: We suggest rs7527939 to be the strongest indicator of susceptibility to schizophrenia in the Bulgarian population within the HHAT locus.

Application of directly coupled LC-NMR-MS to the structural elucidation of metabolites of the HIV-1 reverse-transcriptase inhibitor BW935U83.

The human in vivo metabolism of the HIV-1 reverse transcriptase inhibitor 5-chloro-1-(2',3'-dideoxy-3'-fluoro-erythro-pentofuranosyl)uracil (BW935U83) was studied using 19F NMR spectroscopy, directly coupled LC-NMR and LC-NMR-MS. The number and relative proportions of the drug metabolites were obtained from 19F NMR spectra of whole human urine. The novel use of the continuous-flow 19F detected LC-NMR experiment yielded chromatographic retention times and 19F chemical shifts for the parent drug, the glucuronide conjugate of the parent and an early eluting polar metabolite. The parent drug and its glucuronide conjugate were easily characterised by directly coupled 1H LC-NMR spectroscopy and two-dimensional TOCSY experiments. The identification of the second metabolite was achieved using 19F NMR and directly coupled 1H LC-NMR-MS which furnished the molecular weight, and through the use of MS-MS techniques, information on the fragment ions. This species was identified as 3-fluoro-ribolactone.

Safety and pharmacokinetics of 5-chloro-2',3'-dideoxy-3'-fluorouridine (935U83) following oral administration of escalating single doses in human immunodeficiency virus-infected adults.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83) is a nucleoside analog reverse transcriptase inhibitor that has demonstrated selective anti-human immunodeficiency virus (HIV) activity in vitro and favorable safety profiles in monkeys and mice. A phase I study was conducted to evaluate the safety and pharmacokinetics of six escalating single oral doses of 935U83 in 12 HIV-infected adults. The effect of a high-fat meal on the oral bioavailability of 935U83 was also assessed. The volunteers enrolled had CD4+ cell counts ranging from < 50 to 753 cells per mm3 (median, 198). In the dose range of 100 to 1,500 mg 935U83 was well tolerated by all subjects with no drug-related adverse events reported. No significant clinical or laboratory abnormalities were observed throughout the study. 935U83 was rapidly and well absorbed following oral administration with peak plasma concentrations (Cmax) occurring at 0.8 to 1.3 h postdosing. Mean Cmax and AUC0-infinity values of 935U83 were nearly dose proportional in the 100- to 1,500-mg dose range (from 2.4 to 30 micrograms/ml and from 3.4 to 59 h.micrograms/ml, respectively). Elimination of 935U83 from the plasma was rapid, with an apparent half-life of 1.3 to 1.7 h which was independent of the dose level. Administration of 935U83 with a high-fat meal decreased the rate of 935U83 absorption (mean Cmax decreased by approximately 50% and mean time to Cmax increased by approximately 1 h) but did not affect the extent of oral bioavailability (AUC0-infinity) of 935U83. The 5'-O-glucuronide conjugate was the principal metabolite of 935U83 and was present in the plasma of all volunteers at concentrations lower than 935U83. The molar AUC0-infinity ratio (935U83 glucuronide to the parent compound) was similar across all dose levels (mean, 21 to 27%). At least 60% of the 935U83 dose was absorbed, and approximately an equal percentage of the dose (approximately 30%) was excreted as unchanged 935U83 and as 935U83 glucuronide. Systemic exposure to 935U83 at levels exceeding its average in vitro antiretroviral 50% inhibitory concentration (approximately 0.5 microgram/ml or 1.8 microM) can be achieved after a single oral dose.

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2',3'-dideoxy-3' -fluorouridine (935U83) in human plasma.

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2',3'-dideoxy-3'-fluorouridine (935U83; I) directly and its 5'-glucuronide metabolite (5-chloro-2',3'-dideoxy-5'-O-beta-D-glucopyranuronosyl-3'-fluorour idine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with beta-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from -5.5 to 7.1% during the validation and from -6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-microliters sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.

Intrathecal melphalan therapy of human neoplastic meningitis in athymic nude rats.

We report the activity and toxicity of intrathecal melphalan in the treatment of human neoplastic meningitis in the subarachnoid space of athymic nude rats. Animals received injections via chronic indwelling subarachnoid catheters with 5 x 10(5) or 5 x 10(6) TE-671 human rhabdomyosarcoma cells or 5 x 10(6) D-54 MG human glioma cells and were treated with melphalan on days 8, 5, or 5, respectively. Melphalan toxicity in nontumor-bearing rats was assessed at single doses of a 2.0, 3.0, 4.0, or 5.0 mM solution, with clinical and histological evidence of neurotoxicity observed at the 4.0 and 5.0 mM levels. Multiple-dose toxicity studies using a dosing schedule of twice a week for two weeks with a 0.25, 0.5, 0.75, 1.0, 1.5, or 2 mM solution revealed dose-dependent clinical and histological evidence for toxicity at all dosages. Treatment of TE-671 with a single dose of 2.0 mM intrathecal melphalan produced an increase in median survival of 442% compared with saline controls (P < 0.003). Comparison of a single dose of 1.0 or 2.0 mM melphalan with a multiple dose regimen at 0.25 or 0.5 mM melphalan in the treatment of TE-671 revealed increases in median survival of 50% for 1.0 mM, 57% for 2.0 mM, 79% for 0.5 mM, and 111% for 0.25 mM concentrations. Comparison of a single dose of 1 mM melphalan with multiple doses of 0.25 mM melphalan in the treatment of D-54 MG revealed an increase in median survival of 475+% for each of the regimens. Intrathecal melphalan may be an important new addition in the treatment of neoplastic meningitis and is currently being evaluated clinically in a Phase 1 trial.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83), a selective anti-human immunodeficiency virus agent with an improved metabolic and toxicological profile.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83) is a selective anti-human immunodeficiency virus (HIV) agent. When tested in phytohemagglutinin-stimulated normal human peripheral blood lymphocytes against fresh clinical isolates of HIV type 1 (HIV-1) obtained from patients naive to AZT (3'-azido-3'-deoxythymidine [zidovudine]), 935U83 inhibited virus growth with an average 50% inhibitory concentration (IC50) of 1.8 microM; corresponding IC50s were 0.10 microM for FLT (3'-deoxy-3'-fluorothymidine) and 0.23, 0.49, and 0.03 microM for the approved agents AZT, ddI (2',3'-dideoxyinosine), and ddC (2',3'-dideoxycytosine), respectively. Importantly, 935U83 retained activity against HIV strains that were resistant to AZT, ddI, or ddC. Of additional interest, we were unable to generate virus which was resistant to 935U83 by passaging either HXB2 (AZT-sensitive) or RTMC (AZT-resistant) strains in the presence of high concentrations of 935U83. The anabolic profile of 935U83 was similar to that of AZT, and 935U83 triphosphate was a potent inhibitor of HIV-1 reverse transcriptase. Pharmacokinetic evaluation showed good oral bioavailability (86% in mice and 60% in monkeys) and less extensive metabolism to the glucuronide relative to AZT. 935U83 showed low toxicity. In an in vitro assay for toxicity to a human erythrocyte progenitor, erythroid burst-forming unit (BFU-E), the IC50 for 935U83 (> 400 microM) was more than 1,000-fold those of FLT (0.07 microM) and AZT (0.30 microM). Mild reversible reductions in erythrocytes and associated parameters were seen in mice dosed orally with 2,000 mg of 935U83 per kg per day for 1 and 6 months. In monkeys dosed orally with up to 700 mg/kg/day for 1 and 6 months, the only possible treatment-related finding was cataracts in 1 of 12 animals given the intermediate dose of 225 mg/kg/day. At the highest doses in mice and monkeys, maximal concentrations in plasma were more than 100-fold the anti-HIV IC50s against clinical isolates. This safety profile in animals compares very favorably with that of any of the anti-HIV drugs approved to date and has led us to begin evaluation of 935U83 in patients with HIV infection.

Probenecid-impaired biliary excretion of acetaminophen glucuronide and sulfate in the rat.

Acetaminophen (APAP; 100 mg/kg iv) and probenecid (50 mg/kg bolus + 11.4 mg/hr/kg infusion) were administered to male Sprague-Dawley rats to examine the disposition of APAP, and its glucuronide (AG) and sulfate (AS) conjugates in plasma, bile, and urine. Probenecid significantly decreased the formation clearance of AG from 3.65 +/- 0.434 to 1.94 +/- 0.441 ml/min/kg and the renal clearance of AS from 9.32 +/- 2.26 to 3.15 +/- 1.21 ml/min/kg. The biliary excretion of AG was reduced approximately 3- to 4-fold by probenecid, from 6.54 to 1.87% of the APAP dose, and the AG biliary excretion rate was decreased 4- to 5-fold during probenecid treatment. The more extensive impairment of AG biliary excretion relative to AG formation suggests that probenecid may inhibit the hepatobiliary transport of AG. The significant reduction in the biliary excretion rate at early time points for AS suggests that probenecid may inhibit hepatic AS transport. The study results indicate that probenecid impairs AG and AS formation, AS renal secretion, and AG and AS biliary excretion.

Hyperthermia-induced enhancement of melphalan activity against a melphalan-resistant human rhabdomyosarcoma xenograft.

The effects of regional hyperthermia (42 degrees C for 70 min) on the antitumor activity of melphalan were examined in athymic mice bearing melphalan-resistant human rhabdomyosarcoma (TE-671 MR) xenografts growing in the right hind limb, and results were compared with similar studies of melphalan-sensitive (TE-671) parent xenografts. Melphalan alone at a dose of 36 mg/m2 (0.5 of the 10% lethal dose) produced growth delays of 4.1 to 10.2 days in TE-671 MR xenografts and 21.8 to 28.7 days in TE-671, respectively. Hyperthermia alone produced growth delays of 0.9 days in TE-671 MR xenografts and 0.8 days in TE-671. Combination therapy with melphalan and hyperthermia produced growth delays of 7.2 to 13.3 days in TE-671 MR xenografts and 34.3 to 42.8 days in TE-671, respectively, representing a mean thermal enhancement ratio of 1.7 in TE-671 MR and 1.5 in TE-671. Measurement of glutathione levels in TE-671 MR xenografts following treatment with melphalan, hyperthermia, or melphalan plus hyperthermia revealed significant reductions in glutathione content with the nadir (60% of control values) seen 6 h following treatment. Glutathione levels in TE-671 xenografts following identical therapy revealed no differences from control values. Hyperthermia plus melphalan did not result in a higher tumor-to-plasma melphalan ratio compared with treatment with melphalan alone in either TE-671 MR or TE-671 xenografts. These studies suggest that heat-induced alterations in tumor glutathione or melphalan levels are not responsible for the increase in melphalan activity produced by hyperthermia. Combination therapy with melphalan plus regional hyperthermia offers promise for treatment of melphalan-resistant neoplasms.

Therapeutic analysis of melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR.

Investigations with the melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR were carried out to identify patterns of cross-resistance and collateral sensitivity and to define the mechanism(s) mediating melphalan resistance. TE-671 MR was cross-resistant to thio-TEPA, mitomycin, vincristine, and cisplatin, and partially resistant to chlorambucil and cyclophosphamide. TE-671 MR and the parent line TE-671 were both resistant to 1,3-bis(2-chloroethyl)-nitrosourea and expressed similar levels of O6-alkylguanine-DNA alkyltransferase. TE-671 MR retained full sensitivity to actinomycin D and demonstrated enhanced sensitivity to VP-16 compared to TE-671. Treatment of TE-671 MR with melphalan plus VP-16 resulted in greater than additive growth delays. The frequency of hypoxic regions was similar in TE-671 MR and TE-671, respectively. Measurement of tumor-to-plasma levels at 180 min following i.p. administration of melphalan at 0.5 of the 10% lethal dosage showed mean tumor-to-plasma ratios of 3.81 in TE-671 MR and 7.38 in TE-671, respectively. The lower drug levels in TE-671 MR may be contributing to the resistance to melphalan and thus indicate the need for further studies to define the reasons for these differences in tumor drug level.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

Optimal perfusion rate determined for in situ intestinal absorption studies in rats.

Iopanoic acid was used as a model compound to study the effect of the intestinal perfusion rate on the mean absorption clearance. Absorption of iopanoic acid followed first-order kinetics, with a first-order absorption rate constant (ka) linearly dependent on the dry intestinal weight. An absorption clearance--time plot revealed three phases. Phase I represented an equilibration phase, Phase II was a uniform phase, and Phase III was a physiological deterioration of the animal under prolonged anesthesia. The variability in the observations during Phase II of the absorptive clearance--time profiles was assessed statistically, and the minimum occurred at 9.9 microliters/sec (0.594 ml/min). The relation between the coefficient of variance (CV) and the perfusion rate is given by CV = (-5.52 X 10(-5)Q3 + (2.78 X 10(-3)Q2 - (3.87 X 10(-2)Q + 0.243, where Q is the perfusion rate through the intestinal lumen. These studies demonstrate that an optimal flow rate exists for minimizing the variability in in situ absorption studies. The dependency of the absorption clearance on the intestinal perfusion rate appears to conform to the convective diffusion model.