Isotope-specific analysis of Ni by ICP-MS: applications of stable isotope tracers to biokinetic studies.

Inductively coupled plasma mass spectrometry (ICP-MS) offers excellent detection limits and isotopic analysis of Ni in aqueous standards, but is prone to interferences--mainly from Ca-containing polyatomics--when biological matrices are analyzed for Ni. We have used multivariate calibration with principal components analysis (PCA) to correct for mass overlaps in serum digests. The resulting detection limit for Ni is below 1 microgram/l and the within-run imprecision is 6% at 1.46 micrograms Ni/l. In urine, the higher Ca content renders routine application of PCA problematic. We evaluated several methods of pre-concentration, and have developed a method of Ca oxalate precipitation that allows direct analysis of Ni in the diluted supernatant. The stable isotope 62Ni and the radiosotope 63Ni were co-administered i.v. to rats and the serum and urinary clearances were determined by liquid scintillation counting and ICP-MS. Ni measurements by both methods were in excellent agreement, and serum clearance fit a double exponential decay consistent with the two-compartment model of Onkelinx et al. [24]. A human volunteer ingested 61Ni (20 micrograms Ni/kg body wt.) in water after an overnight fast. Identical serum levels, peaking near 35 micrograms/l at 2 h, were measured by electrothermal atomic absorption spectrometry and ICP-MS with PCA. Urinary excretion of 61Ni measured by ICP-MS demonstrated absorption of 30% of the administered dose. We conclude that Ni isotopes can be measured in body fluids by ICP-MS at levels that allow stable isotope tracer studies in humans.

Speciation of tissue and cellular iron with on-line detection by inductively coupled plasma-mass spectrometry.

Iron accumulating to excess in tissues of humans and animal models occurs mainly as complexes with transferrin, ferritin, other hemoproteins, and insoluble hemosiderin particles. To determine the distribution of Fe amongst these molecular species, we have used inductively coupled plasma-mass spectrometry as a means of on-line, isotope-specific detection for their liquid chromatographic separation. The stable isotope 57Fe is a suitable isotope for monitoring the Fe content of each fraction, and its availability at high isotopic enrichment makes it an attractive choice for tracer studies when the use of a radioisotope is undesirable, e.g., in human subjects. The detection system offers the advantages of high sensitivity (detection limits in the parts per billion range), a wide dynamic range (linearity of the calibration curve over several orders of magnitude), and on-line analysis facilitating real-time evaluation of the chromatographic separation, in addition to isotope-specific information. The Fe distributions in healthy rat livers, liver and heart tissue from Fe-loaded human subjects, and human hepatocyte cultures are reported. The ferritin:hemosiderin ratio in these samples is shown to be an indicator of the degree of Fe loading and correlates well with that determined by Zeeman-corrected electrothermal atomic absorption as an alternative means of detection.

Liposomes as cyclosporin A carriers: ESR study of cyclosporin A interaction with 1,2-dimyristoyl-sn-3-phosphatidylglycerol liposomes.

In order to study the interaction of cyclosporin A (CsA) with phosphatidylglycerol liposomes, thermally induced changes of 1,2-dimyristoyl-sn-3-phosphatidylglycerol (DMPG) liposomes and DMPG liposomes containing CsA were studied by electron spin resonance (ESR) spectroscopy using 5-, 7-, 10-, 12- or 16-nitroxide stearic acid (NO-SA) as spin probes. All liposomes were purified by Sepharose 4B column chromatography and the molar ratio of [14C]DMPG/[3H]CsA in these liposomes was found to be 31.1 +/- 0.6. ESR spectroscopy established that the gel-to-liquid crystalline phase transition of DMPG liposomes occurred sharply at 22 degrees C, shifted significantly to 26-34 degrees C in the presence of CsA, detected with 5-, 7-, 10- and 12-NO-SA probes. Furthermore, transitions at 23 degrees C and 31 degrees C were detected with 16-NO-SA probe in the same liposomes containing CsA. It was concluded that CsA interacts with both the polar head group region and the entire hydrocarbon region of DMPG and must be embedded in the hydrophobic region of liposomes.

Liposomes as carriers of cyclosporin A.

Liposomes prepared from 1,2-dimyristoyl-sn-3-phosphatidylglycerol (DMPG) and 1,2-dimyristoyl-sn-3-phosphatidylcholine (DMPC) containing cyclosporin A (CsA) were reviewed in respect of the amount and localization of associated CsA. It was found that DMPG liposomes are capable of binding a much higher amount of CsA than those prepared from DMPC. Furthermore, while CsA associated with DMPG was embedded within the hydrophobic region of phospholipids, CsA associated with DMPC was excluded from the hydrophobic region and was entrapped in the hydrophilic region of these liposomes.

Liposomes as cyclosporin A carriers: the influence of ordering of hydrocarbon chains of phosphatidylglycerol liposomes on the association with and topography of cyclosporin A.

The addition of cholesterol and stearylamine to liposomes prepared from phosphatidylglycerol containing saturated and unsaturated fatty acids was studied to determine the effect on binding of cyclosporin A (CsA). It was found that liposomes containing phosphatidylglycerol-stearylamine-cholesterol in the molar ratio of 7:2.25:2 had the highest capacity to bind CsA (36.7 mol of phospholipid to 1 mol of CsA), while binding of CsA to phosphatidylglycerol liposomes, phosphatidylglycerol-stearylamine liposomes, or phosphatidylglycerolcholesterol liposomes was significantly lower (342.9, 174.4 and 422.3 mol of phospholipid to 1 mol of CsA, respectively). The binding of CsA to all these liposomes was compared with their order parameters, obtained by electron spin resonance spectroscopy using 5-nitroxide stearic acid spin probe. It was found that increased binding of CsA paralleled the increased ordering of hydrocarbon chains achieved by addition of cholesterol to these liposomes. When liposomes were prepared from pure dimyristoylphosphatidylglycerol (DMPG), 30.7 mol of DMPG were required to bind 1 mol of CsA. The topography of CsA in phosphatidylglycerol liposomes was established by using 5-nitroxide stearic acid as a spin probe. In DMPG liposomes, CsA was found in hydrocarbon chains adjacent to the polar head group, while in liposomes prepared from phosphatidylglycerol containing mixed fatty acids, CsA was associated with the polar head group region without penetrating the hydrocarbon chains of phosphatidylglycerol.

Liposomes as carriers of macrolides: preferential association of erythromycin A and azithromycin with liposomes of phosphatidylglycerol containing unsaturated fatty acid(s).

To assess the most favourable phospholipid composition of a liposomal carrier for antibiotics, small multilamellar liposomes were prepared from phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol of varying fatty acid composition in the presence of erythromycin A and azithromycin. Crude liposomes were subjected to Sepharose CL-4B column chromatography, and liposomes containing antibiotics were well separated from free antibiotics. These experiments established that the greatest association of antibiotics was achieved with liposomes prepared from phosphatidylglycerol rather than phosphatidylcholine or phosphatidylethanolamine. Furthermore, the composition of fatty acids in phosphatidylglycerol liposomes influenced the amount of antibiotics associated with liposomes; the highest amount was obtained with dioleoylphosphatidylglycerol followed by phosphatidylglycerol of fatty acid composition similar to that of egg yolk lecithin. It was established that purified liposomes, prepared from [3H]phosphatidylglycerol containing unsaturated fatty acid(s) bind about 25 per cent of originally present antibiotic. Both antibiotics, erythromycin A and azithromycin, were similar in respect to the amount of their association with liposomes. Determination of the size of phosphatidylglycerol/antibiotic liposomes established that the mean diameter of liposomes containing antibiotics was 200-350 nm, very close to that of liposomes without them.

Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochondrial membranes isolated from guinea pig and rat liver.

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.

Biosynthesis of phosphatidylglycerol and phosphatidylglycerolphosphate in submitochondrial membranes isolated from guinea pig liver is absolutely dependent on CDP-diglycerides imported from microsomal membranes.

The biosynthesis of radioactively labelled phosphatidylglycerol via phosphatidylglycerophosphate in outer and inner mitochondrial membranes isolated from guinea pig liver was found to depend absolutely on CDP-diglycerides, which could not be biosynthesized in these membranes. The requirement for CDP-diglycerides in the biosynthesis of labelled phosphatidylglycerol could be fulfilled by the transfer of biosynthesized [3H]CDP-diglycerides from the microsomal membranes to the outer and inner mitochondrial membranes.

Comparison of the biosynthesis and composition of polyglycerophosphatides and phosphatidylinositols in mitochondria and microsomes isolated from neonatal and adult rat heart and liver.

The level of biosynthesis and the composition of polyglycerophosphatides (phosphatidylglycerol, phosphatidyglycerolphosphate, and diphosphatidylglycerol or cardiolipin) and phosphatidylinositols were examined in mitochondria and microsomes, respectively, isolated from neonatal and adult rat heart and liver. Biosynthesis of [3H]polyglycerophosphatides [( 3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate) was 4.5 times higher in neonatal than in adult heart mitochondria, whereas in the respective liver mitochondria this synthesis was only 15% higher in neonatal mitochondria. The biosynthesis of [3H]phosphatidylinositol was twice as high in neonatal as in adult heart microsomes, but very similar in the respective liver microsomes. The major biosynthesized polyglycerophosphatide was [3H]phosphatidylglycerol. The accumulation of [3H]phosphatidylglycerolphosphate depended on the origin of the mitochondria. Under our experimental conditions [3H]phosphatidylinositol was the only synthesized phosphoinositide in all microsomes. The biosynthesis of cardiolipin depended on the origin of the mitochondria and was highest in adult rat liver mitochondria and lowest in adult heart mitochondria. In all cases the biosynthesized [14C,3H] cardiolipin from [14C]phosphatidylglycerol and [3H]CDP-diglycerides had a ratio of 14C/3H around unity. The biosynthesis of [3H]CDP-diglycerides, the key precursor for the biosynthesis of phosphatidylglycerol, phosphatidylinositol, and cardiolipin, was 30% higher in neonatal than in adult heart microsomes and very similar in the respective liver microsomes. The subcellular localization of the enzymes required for the biosynthesis of the lipids and liponucleotides examined was found to be the same in membranes isolated from neonatal and adult rat heart and liver.

The topography of acetylcholinesterase in dimyristoylphosphatidylcholine liposomes.

Unilamellar liposomes prepared from sn-3-(dimyristoyl)phosphatidylcholine (DMPC) in the presence and absence of acetylcholinesterase were examined by ESR for lipid/protein interactions. Using 5-, 12-, and 16-doxyl stearic acid probes incorporated into the phospholipid bilayers, no measurable differences in the gel to liquid-crystalline phase transition temperature of DMPC (as determined by ESR spectroscopy) were observed when the enzyme was present. These results have established that no significant incorporation of acetylcholinesterase into the hydrophobic region of the phospholipid bilayer is detectable at the protein: lipid ratios used in these experiments. Confirmation of these results was also obtained by differential scanning calorimetry. Interaction of the enzyme with the outermost region of the bilayer was established by trypsin digestion which indicated that as much as 25% of liposome-associated enzyme was removable and was, therefore, exposed to the outer surface. The results of this study have established that acetylcholinesterase associated with unilamellar DMPC liposomes was primarily entrapped within the aqueous compartment of the vesicles and was not present in the phospholipid bilayer.

Liposomes as cyclosporin A carriers: positively charged lecithin-cholesterol liposomes associated with cyclosporin A do not inhibit biosynthesis of mitochondrial polyglycerophosphatides and microsomal phosphatidylinositol.

Phosphatidylcholine (from egg yolk)-cholesterol-stearylamine (7:2:2.25, molar ratio) liposomes when associated with cyclosporin A (phosphatidylcholine:cyclosporin A = 2:0.07, molar ratio) do not inhibit significantly the biosynthesis of [3H]polyglycerophosphatides and [3H]phosphatidylinositol in mitochondrial and microsomal membranes, respectively, isolated from rat liver. These results are explained by possible protection of the liposomes by cyclosporin A against phospholipase A2 hydrolysis.

Evidence that the effect of soya bean phosphatidylinositol-cholesterol-cyclosporin liposomes on the biosynthesis of acidic phospholipids in isolated subcellular membranes from rat liver and heart is attributable to the soya bean phosphatidylinositol-cholesterol liposomes.

This study examined the effect of adding soya bean phosphatidylinositol-cholesterol (2:1 molar ratio) liposomes and soya bean phosphatidylinositol-cholesterol (2:1 molar ratio)-cyclosporin liposomes on the biosynthesis of [3H]polyglycerophosphatides (phosphatidylglycerolphosphate, phosphatidyl-glycerol and cardiolipin) and [3H]phosphatidylinositol in mitochondria and microsomes, respectively, isolated from rat liver and heart. The results obtained with liposomes, and liposomes containing cyclosporin, established that cyclosporin did not participate in the metabolism of acidic phospholipids. In rat liver and heart mitochondria the amount of biosynthesized [3H]polyglycerophosphatides fell to one-third and a half respectively in comparison with the amounts found when liposomes were not added. The composition of [3H]polyglycerophosphatides in both mitochondria showed an increased accumulation of [3H]phosphatidylglycerolphosphate with a concomitant decrease of about 30 per cent in [3H]phosphatidylglycerol. The biosynthesis of [3H]phosphatidylinositol in both rat liver and rat heart microsomes fell to one-third of that found in the absence of liposomes. Under similar conditions the biosynthesis of [3H]cardiolipin decreased by 50 per cent in both mitochondria. The incubation of the same liposomes and liposomes containing cyclosporin with isolated rat liver mitochondria and microsomes released about 5-7 per cent of the linoleic acid. When a corresponding amount of linoleic acid was added to the incubation mixture, the biosynthesis of [3H]polyglycerophosphatides in rat liver did not change significantly, whereas in rat heart mitochondria this biosynthesis decreased by about 33 per cent. Under these conditions, the accumulation of [3H]phosphatidylglycerolphosphate and the decrease of [3H]phosphatidylglycerol was established. The biosynthesis of [3H]phosphatidylinositol in the presence of linoleic acid in rat liver and rat heart microsomes was significantly reduced. Under similar conditions the addition of stearic acid did not significantly affect the level or composition of biosynthesized [3H]polyglycerophosphatides or biosynthesized [3H]phosphatidylinositol.

Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides.

The biosynthesis of [3H]polyglycerophosphatides ([3H]phosphatidylglycerophosphate and [3H]phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized [3H]polyglycerophosphatides and the relative amounts of biosynthesized [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-[2-3H]glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized [3H]polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized [3H]polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of [3H]phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of [3H]phosphatidylglycerolphosphate and increased the amount of accumulated [3H]phosphatidylglycerol, but the absolute amounts of these [3H]polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin. The formation of [3H]cardiolipin from [3H]phosphatidylglycerol in the presence of CDP-didecanoin and Mn2+ was found in both the outer and inner mitochondrial membranes.

Effect of treatment of isolated mitochondrial membranes with phosphatidylinositol-cholesterol liposomes on the biosynthesis and composition of polyglycerophosphatides.

Interaction between mitochondria isolated from rat liver and [14C]phosphatidylinositol-cholesterol (2:1 molar ratio) liposomes was studied by following the level of biosynthesis and the composition of mitochondrial [3H]polyglycerophosphatides. After treatment with liposomes, biosynthesis decreased by about 30 per cent and there was a three-fold increase in biosynthesized [3H]phosphatidylglycerolphosphate. The effect of adding proteoliposomes (consisting of mitochondrial protein and liposomes) to mitochondria that had previously been treated with liposomes was also studied in respect to the composition of [3H]polyglycerophosphatides. The results are discussed in relation to the biological membrane-liposome interaction.

Comparison of cytidinediphospho-sn-1,2-diglyceride transfer from microsomal and liposomal to mitochondrial membranes.

Transfer of [3H]CDP-diglycerides from isolated guinea pig liver microsomal and liposomal membranes to guinea pig mitochondrial membranes was studied by incubating microsomal or liposomal membranes carrying [3H]CDP-diglycerides with mitochondrial membranes and determining the CDP-diglyceride-dependent incorporation of sn-3-[14C]glycerolphosphate into mitochondrial [14C]polyglycerophosphatides. A significant difference in the amount of transferred [3H]CDP-diglycerides and the composition of mitochondrial [14C]polyglycerophosphatides was found depending on whether [3H]CDP-diglycerides were transferred from microsomal or liposomal membranes. This amount was around 12% when [3H]CDP-diglycerides were transferred from the microsomal membranes and around 4.6% when they were transferred from the liposomal membranes. Furthermore, about 60% of [14C]phosphatidylglycerol and 35% of [14C]phosphatidylglycerophosphate were found in the microsomes-mitochondria system and about 9% of [14C]phosphatidylglycerol and 79% of [14C]phosphatidylglycerophosphate were found in the liposomes-mitochondria system, establishing an important role for the membrane donor in the transfer of [3H]CDP-diglycerides to mitochondria. Furthermore, if the transfer of [3H]CDP-diglycerides from the microsomal to the mitochondrial membranes was assayed by the determination of [3H]CDP-diglycerides in reisolated mitochondrial membranes without further incorporation into mitochondrial polyglycerophosphates, it amounted to about 38%.

Effect of chlorpromazine on the synthesis, hydrolysis, and transfer of microsomal cytidine liponucleotides and mitochondrial polyglycerophosphatides.

The effect of chlorpromazine on subcellular biosynthesis, hydrolysis, and transfer of lipids and liponucleotides participating in the biosynthesis of polyglycerophosphatides in guinea pig liver was studied. Chlorpromazine showed an apparent stimulation of accumulation of phosphatidic acid and CDP-diglycerides in microsomal membranes and phosphatidylglycerolphosphate in mitochondrial membranes in a concentration-dependent manner that was influenced by incubation time and the nature of fatty acids in CDP-diglycerides. Transfer of membrane-bound CDP-diglycerides from microsomal to mitochondrial membranes was established by the CDP-diglyceride-dependent biosynthesis of phosphatidylglycerolphosphate and phosphatidylglycerol and appeared to be inhibited by the addition of chlorpromazine by about 20%. Evidence was obtained for the formation of a molecular complex between phosphatidic acid and chlorpromazine; this was thought to be responsible for the protection from phosphatidate phosphohydrolase at the concentrations of chlorpromazine and Mg2+ examined.

Co-encapsulation of cyclosporin and insulin by liposomes.

Evidence for an association of both hydrophilic insulin and hydrophobic cyclosporin with liposomes prepared from egg yolk lecithin, cholesterol, and stearylamine (7:2:2.25 molar ratio) was obtained by Sepharose-4B gel filtration. The method used to prepare unilamellar liposomes containing 29.7 nmol cyclosporin and 2.3 nmol insulin per mu mol of liposomal lecithin is described.

Aggregation of unilamellar lecithin liposomes induced by cyclosporin.

Small unilamellar [14C]lecithin liposomes prepared in the presence of cyclosporin sedimented at 12,000 g. Sepharose 4B gel filtration of the resuspended pellet and supernatant yielded identical peaks consisting of small unilamellar liposomes containing cyclosporin. The column retained 40-50 per cent of 14C-labelled liposomes prepared in the presence of cyclosporin as liposome aggregates.

Participation of the microsomal CDP-diglycerides in the mitochondrial biosynthesis of phosphatidylglycerol.

Participation of microsomal CDP-diglycerides in mitochondrial biosynthesis of phosphatidylglycerol was studied by [3H]palmitoyl, [14C]linoleoyl, and [14C]arachidonoyl CDP-diglycerides and [3H]CDP-diglycerides which were bound to microsomal membranes, incubated with unlabelled mitochondrial membranes, and further incubated in the presence of radioactive sn-glycero-3-phosphate under conditions required for mitochondrial phosphatidylglycerol biosynthesis. Ten to 15% of microsomal radioactive CDP-diglycerides was transferred to mitochondrial membranes and incorporated into mitochondrial radioactive lipids identified as phosphatidylglycerol, phosphatidylglycerophosphate, and, when [14C]linoleoyl CDP-diglycerides were used, diphosphatidylglycerol (cardiolipin).