On the validity of fluorimetric intracellular calcium detection: Impact of lipid components.

We investigated the effects of different lipids on the activity of the angiotensin II type 1 receptor (AT1R). As calcium plays a key role in the signaling of the AT1R, we used the calcium-sensitive fluorescence indicators fura-2 to detect intracellular calcium release upon stimulation with the agonist angiotensin II. At first sight, cells preincubated with Very low-density lipoprotein (VLDL) showed a reduced calcium release triggered by angiontensin II compared to untreated control. However, on closer examination, this result seemed to be an artifact. Incubation with VLDL reduced also the amount of intracellular fura-2, as measured by fluorescence in the isosbestic point. Additionally, the maximal obtainable ratio, obtained after complete saturation with calcium ions, was reduced in cells preincubated with VLDL. These findings rendered our initial results questionable. We report the results of our work and our suggestions regarding the experimental setup to contribute to the understanding of the interpretation of fura-2 measurements and to avoid erroneous conclusions.

Lipoprotein turnover and possible remnant accumulation in preeclampsia: insights from the Freiburg Preeclampsia H.E.L.P.-apheresis study.

BACKGROUND: Preeclampsia is a life-threatening disease in pregnancy, and its complex pathomechanisms are poorly understood. In preeclampsia, lipid metabolism is substantially altered. In late onset preeclampsia, remnant removal disease like lipoprotein profiles have been observed. Lipid apheresis is currently being explored as a possible therapeutic approach to prolong preeclamptic pregnancies. Here, apheresis-induced changes in serum lipid parameters are analyzed in detail and their implications for preeclamptic lipid metabolism are discussed. METHODS: In the Freiburg H.E.L.P.-Apheresis Study, 6 early onset preeclamptic patients underwent repeated apheresis treatments. Serum lipids pre- and post-apheresis and during lipid rebound were analyzed in depth via ultracentrifugation to yield lipoprotein subclasses. RESULTS: The net elimination of Apolipoprotein B and plasma lipids was lower than theoretically expected. Lipids returned to previous pre-apheresis levels before the next apheresis even though apheresis was repeated within 2.9 ± 1.2 days. Apparent fractional catabolic rates and synthetic rates were substantially elevated, with fractional catabolic rates for Apolipoprotein B / LDL-cholesterol being 0.7 ± 0.3 / 0.4 ± 0.2 [day- 1] and synthetic rates being 26 ± 8 / 17 ± 8 [mg*kg- 1*day- 1]. The distribution of LDL-subclasses after apheresis shifted to larger buoyant LDL, while intermediate-density lipoprotein-levels remained unaffected, supporting the notion of an underlying remnant removal disorder in preeclampsia. CONCLUSION: Lipid metabolism seems to be highly accelerated in preeclampsia, likely outbalancing remnant removal mechanisms. Since cholesterol-rich lipoprotein remnants are able to accumulate in the vessel wall, remnant lipoproteins may contribute to the severe endothelial dysfunction observed in preeclampsia. TRIAL REGISTRATION: ClinicalTrails.gov, NCT01967355 .

Comparison of double-filtration plasmapheresis (DFPP) versus heparin-mediated extracorporeal LDL-precipitation (HELP)-apheresis in early-onset preeclampsia.

OBJECTIVES: Preeclampsia (PE) is a major cause of maternal and fetal mortality, and preterm birth. Previous studies indicate that lipid-apheresis may prolong pregnancy, namely heparin-mediated extracorporeal LDL-precipitation (HELP)- and dextran sulfate cellulose (DSC)-apheresis. We now report on double membrane plasmapheresis (DFPP) in early-onset preeclampsia (eoPE). STUDY DESIGN: Open pilot study assessing the prolongation of pregnancy in PE by lipoprotein-apheresis (DRKS00004527). Two women with eoPE were treated by DFPP and compared to a historical cohort of 6 patients with eoPE treated by HELP-apheresis (NCT01967355). MAIN OUTCOME MEASURES: Clinical outcome of mothers and babies and prolongation of pregnancies (time of admission to birth). RESULTS: Patient 1 (33y; 22 + 5/7GW) received 4 DFPP. Delivery day 19; birthweight 270 g; weight at discharge 2134 g on day 132. Patient 2 (35y; 21 + 4/7GW) received 2 DFPP. Delivery day 19; birthweight 465 g; weight at discharge 2540 g on day 104. DFPP was well tolerated by both patients. CONCLUSIONS: DFPP proved to be save and pregnancies remained stable as long as 19 days. Although babies were born very preterm both babies could finally be dismissed from hospital. No relevant clinical differences between DFPP and HELP-apheresis could be observed. Therefore, DFPP may extend the range of available apheresis techniques to prolong pregnancies in early-onset preeclampsia. However, further studies are necessary to gain more information. REGISTER: (DRKS00004527).

LDL retention time in plasma can be -based on causation- estimated by the lipid composition of LDL and other lipoproteins.

INTRODUCTION: Information on LDL's dynamic behaviour of LDL (i.e. production rate and fractional catabolic rate) are of interest if pathologies, lipid-lowering strategies or LDL-metabolism itself are investigated. Determination of these rates is costly and elaborate. Here we studied the interrelationship of LDL mass, its composition and other lipoproteins. Based on this data, we deducted information about LDL's dynamic behaviour. METHODS: Lipoprotein profiles of n = 236 participants are evaluated. Plasma was separated by sequential ultracentrifugation into VLDL, IDL, LDL and HDL. Additionally, LDL and HDL were separated into subfractions. Stepwise multiple linear regressions were used to study LDL's ApoB mass and lipid composition. Relying on these results and on causation, we constructed a mathematical model to estimate LDL's retention time. RESULTS: The ApoB mass in LDL correlated best among all measured parameters (including corresponding lipid compositions but using no LDL-associated parameters) with the cholesterol ester content in IDL. TG/CE ratios in LDL's subfractions were strongly correlated with the corresponding ratios in IDL and HDL. The constructed mathematical model links the TG/CE ratio of LDL and HDL to LDL's ApoB concentration and enables a good estimate of LDL's retention time in plasma. DISCUSSION: Relying on our statistic evaluations, we assume that i) the production of nascent LDL via IDL as well as ii) LDL's prolonged retention are mapped by the TG/CE ratio in LDL subfractions. HDL's TG/CE ratio is associated with the change in LDL's TG/CE ratio during its retention in plasma. Our mathematical model uses this information and enables-by relying on causation- a good estimation of LDL's retention time.

Apheresis as emerging treatment option in severe early onset preeclampsia.

Based on an early suggestion by Winkler et al. 2003 and a subsequent successful study by Wang et al. 2006 using lipid apheresis (LA) in 9 patients with preeclampsia to prolong pregnancies, the use of apheresis as therapeutic option in severe early onset preeclampsia has received increasing attention. Further studies using different LA systems also prolonged pregnancy and have been published in the last few years. Albeit using different LA systems and relying on different working hypothesis, all studies demonstrated a promising stabilisation against the disease's progression. Overall time from hospitalisation to the need for mandatory delivery was longer for those patients receiving apheresis compared to historical or matched control patients not receiving apheresis. These data will be reviewed and different hypotheses about the beneficial mechanism of action of apheresis will be discussed. Since up to now there is no curative treatment for preeclampsia other than observation and delivery, future work shall be encouraged.

Concomitant intrauterine growth restriction alters the lipoprotein profile in preeclampsia.

OBJECTIVE: Preeclampsia and intrauterine growth restriction (IUGR) are related conditions. We aimed to characterise common lipid changes. METHODS: Triglyceride and cholesterol levels of patients 24-42 weeks of gestation with IUGR (n = 52), hypertensive IUGR (HIUGR, n = 28), and preeclampsia without IUGR (PE, n = 56) were compared to a control group (CTRL, n = 167). In addition, 60 sera (n = 10 of each pathology IUGR, HIUGR, PE (without IUGR) compared to n = 30 matched CTRL) of severe early onset cases <34 weeks of gestation were chosen and further analysed by ultracentrifugation lipid subfractionation including VLDL, IDL, LDL, and HDL composition. RESULTS: In the full cohort we found low cholesterol in IUGR (p = 0.0405), while triglyceride levels were high in PE (p < 0.0001). Lipid concentrations in HIUGR did not differ significantly from CTRL. In the 60 patients analysed by lipid subfractionation, triglyceride levels were increased in the VLDL subfraction in PE (p < 0.01), however, LDL-bound ApoB and cholesterol levels were lower in IUGR and HIUGR (p < 0.0001 for total cholesterol and p < 0.001 for ApoB in both groups), but not in PE when compared to CTRL. CONCLUSION: Low cholesterol, especially LDL cholesterol levels are a feature of IUGR while high triglyceride levels are a feature of preeclampsia. Increased VLDL-triglycerides suggest a disturbed conversion to LDL in preeclampsia. Of note, the presence of IUGR in hypertensive disorders further alters lipid profiles, which may explain heterogeneous data on lipid values for preeclampsia in the literature. Study groups have to be selected carefully to avoid misinterpretation.

Optimizing Antitumor Efficacy and Adverse Effects of Pegylated Liposomal Doxorubicin by Scheduled Plasmapheresis: Impact of Timing and Dosing.

BACKGROUND: Nanoscale drug delivery systems accumulate in solid tumors preferentially by the enhanced permeation and retention effect (EPR-effect). Nevertheless, only a miniscule fraction of a given dosage reaches the tumor, while >90% of the given drug ends up in otherwise healthy tissues, leading to the severe toxic reactions observed during chemotherapy. Once accumulation in the tumor has reached its maximum, extracorporeal elimination of circulating nanoparticles by plasmapheresis can diminish toxicities. OBJECTIVE: In this study, we investigated the effect of dosing and plasmapheresis timing on adverse events and antitumor efficacy in a syngeneic rat tumor model. METHODS: MAT-B-III cells transfected with a luciferase reporter plasmid were inoculated into female Fisher rats, and pegylated liposomal doxorubicin (PLD) was used for treatment. Plasmapheresis was performed in a discontinuous manner via centrifugation and subsequent filtration of isolated plasma. RESULTS: Bioluminescence measurements of tumor growth could not substitute caliper measurements of tumor size. In the control group, raising the dosage above 9 mg PLD/kg body weight did not increase therapeutic efficacy in our fully immunocompetent animal model. Plasmapheresis was best done 36 h after injecting PLD, leading to similar antitumor efficacy with significantly less toxicity. Plasmapheresis 24 h after injection interfered with therapeutic efficacy, while plasmapheresis after 48 h led to fewer side effects but also to increased weight loss. CONCLUSION: Long-circulating nanoparticles offer the unique possibility to eliminate the excess of circulating particles after successful accumulation in tumors by EPR, thereby reducing toxicities and likely toxicity-related therapeutic limitations.