Whole-body R2∗ mapping to quantify tissue iron in iron storage organs: reference values and a genotype.

AIM: To define reference values for the transverse relaxation rate (R2∗) in iron storage organs and to investigate the role of human haemochromatosis protein (HFE) genotype on iron storage. MATERIALS AND METHODS: Whole-body magnetic resonance imaging (MRI) including a five-echo gradient-echo sequence was performed in 483 volunteers (269 men, mean age 59.3 ± 12.2 years) without clinical evidence of an iron storage disease at 1.5 T. R2∗ values were assessed for liver, spleen, pancreas, heart, bones, and brain parenchyma. The HFE genotype was determined regarding the single nucleotide polymorphisms (SNPs) rs74315324, rs1799945, rs41303501, rs1800562, rs1800730. R2∗ values were compared among participants without and with at least one mutation. R2∗ reference values were defined using volunteers without any mutation. RESULTS: Three hundred and one participants had no mutations in any HFE SNP, 182 had at least one mutation. HFE gene mutations were distributed as (heterozygous/homozygous) rs1799945:132/9, rs1800562:33/1, and rs1800730:11/0. Mean R2∗ values ± SD (per second) in the group without mutation were: liver: 33.4 ± 12.7, spleen: 24.1 ± 13.8, pancreas: 27.2 ± 6.6, heart: 32.7 ± 11.8, bone: 69.3 ± 21.0, brain parenchyma: 13.9 ± 1.2. No significant difference in R2∗ values were found between participants with and without the HFE gene mutation for any examined iron storage organ (pliver=0.09, pspleen=0.36, ppancreas = 0.08, pheart = 0.36, pbone = 0.98, pbrain=0.74). CONCLUSION: Reference values of R2∗ in iron storage organs are feasible to support the diagnosis of iron storage diseases. Non-specific mutations in HFE SNPs appear not to affect the phenotype of tissue iron accumulation.

[The clinical spectrum of urea cycle defects in adult patients]. / Das klinische Spektrum von Harnstoffzyklusdefekten im Erwachsenenalter.

Urea cycle defects belong to the most common metabolic disorders with a cumulative incidence of 1:8000. A common trait of urea cycle defects is a disturbed detoxification of ammonia leading to hyperammonemia in the event of a high nitrogen load. Most patients develop symptoms in the neonatal period or in infancy, e. g. vomiting, seizures and disturbed consciousness. Depending on the affected enzyme and its residual activity, patients differ in the age at first presentation, the character and severity of symptoms and in the susceptibility to metabolic derangement. The presence of hyperammonemia and an altered plasma amino acid profile give the essential diagnostic clues. Since modern therapeutic measures have prolonged the life expectancy of these patients and provided the possibility of a first presentation in adulthood, patients with urea cycle defects have become an increasing challenge in internal medicine. The reported case series illustrates the heterogeneous clinical course of these disorders from childhood to adulthood.

Low trough levels of tipranavir in a combination antiretroviral therapy of tipranavir/ritonavir and tenofovir require therapeutic drug monitoring.

The new non-peptidic protease inhibitor tipranavir is used boosted with ritonavir in a 500/200 mg bid scheme. Multiple drug interactions are described for both drugs because of their different action in CYP450 3A4 and p-glycoprotein. In this retrospective analysis of 22 patients during therapy with tipranavir/ritonavir (TPV) 500 mg/200 mg bid, we found significantly decreased TPV-trough levels in combination with tenofovir (15.32+/-5.22 microg/ml) in comparison to TPV trough levels without tenofovir (20.21+/-14.87 microg/ml). Therapeutic drug monitoring of TPV is recommended.

Safety of long-term lopinavir plasma-levels in patients with liver disease.

Chronic liver disease is often found in HIV infected patients. LPV as first choice drug is often used over long time periods. TDM as a tool in patients care is important but the knowledge of LPV-plasma-levels in patients with chronic liver disease remain uncertain. With this retrosepective analysis we want to show if there are differences in LPV-plasma-levels between patients with and without chronic liver diseases over a long-time period. LPV-plasma-levels were analysed with an HPLC-based methode. The LPV-plasma-levels over the time course in patients with chronic liver disease (n = 30) and patients without liver disease (n = 38) was evaluated. Liver function tests, CD4-cell count and HI-viral load was also correlated with liver disease. The LPV plasma-levels of n = 450 samples from 30 patients with liver disease (Hepatitis B: n = 17, Hepatitis C: n = 16, Alcoholic liver disease: n = 7) were determined over 18.7 +/- 16,3 months (1 - 48.5 months). A median of 10 samples per patient was eligible (2 - 50 samples). There are no significant differences according to liver disease in LPV-plasma levels (mean Ctrough without: 5917 +/- 4811 ng/ml, mean Ctrough with liver-disease: 6564 +/- 4517 ng/ml, p > 0.05). The intraindividual and interindividual variation of LPV-plasma levels, CD-4 increase, HI-virus suppression and liver tests in patients with and without liver disease is comparable. In this clinical setting no differences in LPV-plasma levels between patients with and without chronic liver disease could be demonstrated. LPV-therapy in patients with chronic liver disease is therefore safe. In patients with impaired liver function TDM is a helpful tool for dose adjustment.

High performance liquid chromatographic method for the determination of HIV-1 protease inhibitor tipranavir in plasma of patients during highly active antiretroviral therapy.

A new high-performance liquid chromatographic method for the determination of tipranavir in human plasma is described. Quantitative recovery following liquid-liquid-extraction with diethylether from 100 microl of human plasma was achieved. Subsequently, the assay was performed with 67 mM potassium dihydrogen phosphate-acetonitrile as a mobile phase, a Phenomenex C 18 column and UV detection at 255 nm. Linear Standard curves were obtained for concentrations ranging from 2.5 to 400 microg/ml. The calculated intra- and inter-day coefficents of variation were below 7%.

Follow-up measurements of Nevirapine plasma levels over a prolonged period.

Over a period of more than four years of treatment, 177 Nevirapine plasma levels were taken from 27 patients. The values showed a high inter-patient variability and a lower intra-patient variability. Differences in body weight turned out to be the main reason for inter-patient variability. Treatment over a prolonged period did not result in any change in plasma concentrations. Adjusting dosage by means of therapeutic drug monitoring would appear to be a reasonable way of maximising patient benefit from treatment.

Lipid lowering therapy with fluvastatin and pravastatin in patients with HIV infection and antiretroviral therapy: comparison of efficacy and interaction with indinavir.

BACKGROUND: Lipoprotein disorders in HIV-positive patients receiving highly active antiretroviral therapy (HAART) are becoming a major concern in HIV treatment, since there is growing evidence for an association between HAART-induced hyperlipidemia and increased cardiovascular risk. Yet relatively few data are available on the possible interactions of HAART and treatment with statins. PATIENTS AND METHODS: In this prospective study, 25 HIV-positive, treatment-experienced patients (five female, 20 male, all Caucasian) were treated with either fluvastatin or pravastatin. Total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) levels, and serum triglycerides were determined at regular intervals, as well as therapeutic drug monitoring to assess possible drug interactions. RESULTS: In 13 pravastatin-treated patients, a decrease in total cholesterol levels (from 7.12 mmol/l to 6.29 mmol/l) after 12 weeks of therapy was seen. In 12 patients treated with fluvastatin, a permanent reduction of total cholesterol (from 6.46 mmol/l to 5.31 mmol/l) after 12 weeks was observed. The reduction of LDL levels was 30.2% in the fluvastatin group and 14.4% in the pravastatin group. In eight patients receiving an indinavir-containing HAART, indinavir plasma levels were not significantly influenced. No effect on triglycerides or HDL was observed. CONCLUSION: Fluvastatin and pravastatin are efficient in lowering total and LDL cholesterol levels in HIV-positive patients receiving HAART. Furthermore, no influence on indinavir plasma levels could be observed. Therefore, both compounds seem to be a viable treatment option in HAART-induced hypercholesterolemia.

Liquid chromatographic method for the determination of uridine in human serum.

To evaluate uridine levels in humans we developed a very sensitive and specific high-performance liquid chromatographic method for the determination of uridine in serum. We use techniques which are available in a standard analytical laboratory. Chromatographic analysis was carried out on a Phenomenex Aqua C18 5 micro 125A column protected by a guard cartridge system. Potassium dihydrogen phosphate buffer-acetonitrile was used as an eluent and oxypurinol as the internal standard. All sample preparation steps were done at 4 degrees C and the autosampler was cooled down to 4 degrees C. The calibration curve was linear throughout the calibration range from 0.25 to 100 micromol/l. This method was primarily established to evaluate uridine serum levels in patients with HIV infection since patients on highly active antiretroviral therapy (HAART) might develop metabolic disturbances that could lead to severe and fatal lactic acidosis due to mitochondrial toxicity. It is suggested that a limited or inadequate uridine supply is at least in part responsible for the onset of such deterioration.

Reverse transcriptase-polymerase chain reaction analysis of thyrocyte-relevant genes in fine-needle aspiration biopsies of the human thyroid.

Currently, fine-needle aspiration cytology is a valuable tool in the routine diagnosis of suspicious thyroid nodules. We present a very sensitive method for the molecular analysis of the expression of several genes important for normal thyroid function in parallel to the cytological diagnosis. We adapted reverse transcriptase polymerase chain reaction (RT-PCR) to amplify thyroid-typical mRNAs in samples of thyroid carcinoma cells as small as those obtained by fine-needle aspiration biopsy (FNAB), ie, 100-1000 cells, and applied this procedure to four routinely taken FNABs. Gene products such as thyroglobulin (Tg), thyroid-stimulating hormone-receptor (TSHr), sodium/iodide-symporter (NIS), type I iodothyronine-5'-deiodinase (DI), and type II iodothyronine-5'-deiodinase (DII) were analyzed. To establish RT-PCR protocols, serial dilutions of follicular thyroid carcinoma cells, FTC-133, which express these genes at low levels, were initially used for RNA isolation. Successful RNA isolation and reverse transcription were checked by the amplification of beta-actin mRNA. We detected the mRNAs coding for Tg in as little as 10 cells, for NIS in 100 cells, and for TSHr, DI, and DII in 10,000 cells. After preparing cytological smears of four routinely taken FNABs, all above-mentioned thyroid-typical mRNAs were observed by using the material remaining in the needle for RNA isolation followed by RT-PCR. This method offers the possibility of obtaining two different types of information from the same routinely taken thyroid FNAB: the cytological diagnosis and the expression pattern of several diagnostically relevant genes. Therefore, a more specific diagnosis could be rendered in the preoperative state, and may lead to more specific therapy.

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues.

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [3H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and super-shift assays using a DR2 ("direct repeat" 2) RA response element demonstrated DNA-binding of RARalpha, RARgamma, RXRalpha and RXRbeta in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRalpha1, TRalpha2, and TRbeta. Northern-blot analysis showed low expression of RXRbeta mRNA in FTC-133 and of TRalpha1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta in the 4 cell lines and in human thyroid-carcinoma samples. RARbeta mRNA was reduced in FTC-238 cells and RXRbeta mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding.

Retinoic acid increases sodium/iodide symporter mRNA levels in human thyroid cancer cell lines and suppresses expression of functional symporter in nontransformed FRTL-5 rat thyroid cells.

Decreased iodide uptake in de-differentiated thyroid carcinomas impedes radioiodide therapy. RTPCR analysis revealed reduced expression of Na+/I- symporter (NIS) mRNA in human thyroid carcinomas as compared to normal thyroid. However, in follicular thyroid carcinoma cell lines FTC-133 and FTC-238, treatment with 1 microM all-trans retinoic acid (RA) markedly increased NIS mRNA levels. Anaplastic thyroid carcinoma cell lines HTh74 and C643 showed basal expression of NIS mRNA, but no RA-stimulation. All four cell lines contained the approximately 80 kD NIS protein as judged by Western blot, although they did not accumulate iodide. In contrast, in nontransformed rat FRTL-5 cells, 1 microM RA downregulated NIS mRNA levels, inhibited the TSH- or forskolin-triggered induction of NIS message after TSH-depletion, and reduced iodide uptake to 38% after 5 d. This divergent RA-responsivity of NIS may provide the means to target radioiodide to thyroid carcinomas by upregulating iodide transport into tumor tissue while simultaneously inhibiting iodide accumulation in normal thyrocytes and may thus re-establish the potential for radioiodide therapy.