Selected isotope ratio measurements of light metallic elements (Li, Mg, Ca, and Cu) by multiple collector ICP-MS

The unique capabilities of multiple collector inductively coupled mass spectrometry (MC-ICP-MS) for high precision isotope ratio measurements in light elements as Li, Mg, Ca, and Cu are reviewed in this paper. These elements have been intensively studied at the Geological Survey of Israel (GSI) and other laboratories over the past few years, and the methods used to obtain high precision isotope analyses are discussed in detail. The scientific study of isotopic fractionation of these elements is significant for achieving a better understanding of geochemical and biochemical processes in nature and the environment.
Introduction
The development of multiple collector inductively coupled mass spectrometry (MC-ICP-MS) about 15 years ago immediately indicated the advantages of this technique, at least in ratio measurements from mass 80 and above. In general, the acquired data showed precision equal or better to the well-established thermal ionization mass spectrometry (TIMS) [1, 2]. The ease of data acquisition was remarkable, the ionization efficiency was significantly larger, and consequently the sensitivity was improved. When MC-ICP-MS was introduced it had to compete with TIMS, which at that time was fully developed and provided the best isotope ratio data for metallic elements in terms of precision and accuracy. Even in the stages of application tests MC-ICP-MS exhibited several advantages: high precision, high reproducibility, high analytical throughput, simple sample preparation, and, as mentioned above, improved sensitivity [3].
MC-ICP-MS requires a more complicated instrument compared to TIMS. It uses an ion source at atmospheric pressure and therefore requires a specially designed ion beam introduction inlet system equipped with efficient pumping systems to reduce the argon gas pressure to 10−7−10−8 mbar. Further pumping systems along the ion flight tube maintain the vacuum at 2 × 10−9 mbar. The ion beam emerging from the interface has a circular profile which requires a tunable quadrupole lens, efficiently changing it to a rectangular shape. This rectangular ion beam possesses ions with an energy spread of up to 30 eV. An electrostatic energy filter is used to separate an almost monoenergetic ion beam acceptable for the magnetic sector mass separator. A further inherent problem in an ICP-MS is the relatively high instability of the plasma, observed as fluctuations and drifts of the ion beam. A single collector system, whether a Faraday cup or an electron multiplier, is too slow to follow this instability; therefore an array of Faraday cups (and also a Daly electron multiplier) is mounted at the end of the instrument, allowing the simultaneous monitoring of two or more ion beam intensities. This technique had already been applied in the multiple collector TIMS instruments.
Plasma 54 (P54), the first MC-ICP-MS instrument manufactured by VG Elemental in the UK [1], contained an ion collector equipped with seven Faraday cups and an analog Daly detector. A unique feature of all double focusing instruments is the wide flat-topped ion peak shape, which is of primary importance for high precision data acquisition. Two further modifications were applied to this instrument: a wide-end flight tube to allow simultaneous measurement of ions with large mass differences, such as U–Pb or 6Li–7Li, and the addition of an electrostatic energy filter before the Daly detector to enhance abundance sensitivity measurements. Isotope ratio measurements using laser ablation have also been demonstrated [4]. Several years later VG Elemental-Thermo Elemental introduced the Axiom, a smaller double focusing instrument of vertical configuration, providing high mass resolution capability. This mass spectrometer was aimed for high precision, fast analytical work [5].
Several other MC-ICP-MS instruments were manufactured by other companies applying different approaches for efficient reduction of ionic spectral interferences formed by argon and atmospheric gases, e.g., thermalizing the ions produced by the plasma, introducing a detection system comprising a fixed array of Faraday cups and ion counters, and including an adjustable ion beam dispersion device.
Micromass in the UK introduced the IsoProbe MC-ICP-MS where an off-axis hexapole collision cell is mounted in a jacket between the second interface cone and a beam focusing system [5]. The cell is flushed with a low pressure inert gas which reduces the ion energy to less than 1 eV. Also spectral interferences such as ArC+, ArN+, ArO+, ArCl+, and  dimers produced in the atmospheric plasma, which interfere with 52Cr+, 54Fe+, 56Fe+, 75As+, and 80Se+, respectively, are removed by collision-induced cleavage of the Ar–X bonds, allowing measurement isotope ratios at masses below 80. The remainder of the instrument comprises a magnetic mass separator and a multiple collector detection system.
Nu Instruments in the UK introduced the Nu Plasma MC-ICP-MS. This machine, as with the Plasma 54, was developed by P.A. Freedman who maintained the same basic concepts, but introduced a different design for almost all the modules. Essentially it is a forward geometry (electrostatic sector followed by magnetic sector), double focusing mass spectrometer with a C-shape configuration compared to the S-shape of the P54. The ion beam profile conversion system is of different construction and the electrostatic energy filter and the magnetic separator were changed in shape and size. The profound advance in this design is the multiple collector detection system. The detection module comprises an array of 15 fixed detectors, 12 Faraday cups, and three electron multipliers, in front of which a zoom lens system deflects each separated ion beam into a chosen detector. Also, one of the multipliers is equipped with a filter for cutting off the tail from highly abundant isotopes. A later model from Nu Instruments, the Nu1700, is a large geometry, high resolution MC-ICP-MS [6] that allows one to overcome most of the interferences.
Finnigan MAT in Germany introduced the Neptune MC-ICP-MS, a double focusing C-shape instrument with a large magnet providing 16% mass dispersion, movable collector array, and high resolution capability. Flat-topped peaks are achievable at R > 4,000 [7].
When discussing isotope ratio measurements utilizing plasma ionization, several further phenomena should be mentioned. Isotopic fractionation is of utmost importance. It is caused by repulsive forces in the intensive positive ion beam emerging from the plasma and supersonic ion expansion through the sample cone. Both effects yield radial repulsion of the lighter isotope from the beam center, i.e., increasing the heavy mass over light mass ratio. The fractionation effect is inversely mass dependent from several per mils in uranium to more than 10% in lithium. It is constant in time, since fresh sample solution is continuously aspirated into the ion source. This is in contrast to TIMS ionization, where a fixed sample is used, permanently changing in composition as the lighter isotope is preferentially vaporized. Fractionation may be corrected in one of three techniques: (1) internal and (2) external normalization, including double spike method; and (3) “standard-sample-standard bracketing”; consecutive measurements of the same ratio in a sample and standard. Further details will be given when describing the ratio measurements in the elements discussed in this review.
ICP mass spectrometry is subject to other interferences originating from various sources. Spectral interferences are products of interactions between the carrier gas and atmospheric gases or the solvent molecules. As mentioned above they obscure the lower mass range. Sodium ions in the sample solution in the case of copper analysis may interfere as ArNa+ at mass 63. Further cases are molecular interferences such as oxides, nitrides, and hydrides of trace elements in the solution and isobaric ion interferences. These cases can be removed by using expensive high resolution MS. Other ways to reduce interferences include using of desolvation nebulizer and chromatographic separation of the analyte from the matrix prior to introduction into the MS. This separation is also necessary to prevent the additional effects of mass fractionation connected with the matrix effect. In the case of the heavy isotope ratio measurements, this fractionation is corrected by internal or external normalization, but in the case of standard-sample-standard bracketing, generally used for light masses correction, the analyte must be carefully and precisely separated from the matrix.
As with the stable isotopes of the light elements, the isotopic composition of a sample is given in δ units (‰) relative to standard (I is isotope, x and y are mass numbers).
Generally, the isotope variations are derived from bracketing the measured sample ratio with the mean ratios of a standard measured before (std1) and after the sample run (std2), and are presented as deviations in parts per 1,000 of the measured ratio from that of the standard (normalizing) ratio:
Results and discussion
Lithium
Lithium isotopes are of significant importance in a number of fields, such as geochemistry [8–11], astrophysics [12–13], nuclear technology [14], and biomedicine [15]. MC-ICP-MS opened new frontiers in Li isotope measurements by providing high precision data (<0.3‰, 2σ). Lithium isotopes fractionate during hydrothermal processes, and significant variations may be observed in 7Li/6Li ratios in water derived from marine sedimentary rocks and from hydrothermal altered igneous rocks, thereby providing valuable information regarding regional ground-water flow paths. Lithium isotopes are a powerful tracer of recycling processes in the Earth. This is particularly the case for understanding the geochemical evolution of the Earth’s mantle, because (a) Li is a moderately incompatible constituent of minerals in peridotite, (b) Li isotope ratios show large variations in the terrestrial system, caused by low-temperature fractionation and mixing, (c) Li is a fluid-mobile element. Therefore, combined with existing geochemical information, its isotopes could be powerful geochemical tracers, especially for fluid-related (metasomatic) mantle processes. In recent years, almost all lithium isotope studies dealing with geological and related systems have applied the MC-ICP-MS measurement technique.
Lithium isotope ratio measurements require careful separation of Li from the matrix in natural samples. For solid samples, digestion and ion exchange chromatography separation are used in general; liquid samples are processed by chromatographic separation [16–21]. Acid leaching [22, 23] and direct resin techniques [24] are also applied. The most rapid method of separation was published by Hall et al. [21] for TIMS and modified at the Geological Survey of Israel for MC-ICP-MS. Any traces of Na, K, Mg, Sr, and Rb were detected using this modified method, so the samples were sufficiently pure for ratio measurements by Nu Plasma MC-ICP-MS.
The values of the ratio in standard L-SVEC NBS (NIST, Li2CO3) are  [25], and in seawater  [26]. A typical analysis sequence consists of blank, standard, blank, sample 1, blank, standard, blank, sample 2, and so on. Lithium ratio measurements exhibit time-dependent drifts and random shifts. Tomascak et al. [16] reported a typical 7Li/6Li ratio drift for the L-SVEC standard from 12.7 to 12.3 during an 8-h period. Nishio and Nakai [17] rejected data when two successive measurements of the bracketing standard ratio shifted by more than 2‰. These authors also reported a stability of  (2σ) for an in-house isotope standard measured over an 8-month period. It is good practice to carry out the sample and standard measurements under identical instrumental and solution conditions. The analyte concentrations in both solutions should be as close as possible and the impurity levels identical and as low as possible.
It should be noted that sample purity in TIMS analysis relative to MC-ICP-MS is of major importance, since the large isotopic fractionation in Li is highly sensitive to impurities in the loaded sample, especially when Na and K are interfering elements [21]. It was reported that a Na/Li ratio greater than approximately 5 may cause unstable instrumental fractionation [16]. Moriguti and Nakamura [27] developed a four-step chromatographic separation for Li purification, allowing precise TIMS Li ratio analyses.
Bouman et al. [28] studied a wide range of Li samples originating from islands in the Pacific, Atlantic, and Indian Oceans. Thirty six samples and three standard materials were analyzed. The precision of these measurement are 0.01–0.5(‰), 0.6–1.0(‰), and 1.2–1.9(‰) for 22, ten, and four samples, respectively. The data for standard materials are presented in Table 1, which also summarizes selected MC-ICP-MS Li isotope ratio measurements in geological samples.
Table 1Li isotopic composition in standards and selected natural samples relative to NIST NBS L-SVEC (‰)Sampleδ7Li±2σReferenceJR-2 rock standard3.840.18[24]Magmatic arc lavas (Kurile)[18]8322/3 Onekotan4.2<1.0K33 Keli-Mutu5.1<1.0VB30 granite−1.41.0[19]MG20 granite0.81.0Inorganic calcite CM019−7.60.6[20]Coral Acropora21.00.4Indian Ocean water33.01.2[21]Atlantic Ocean water32.11.2Foraminifera Orbulina universa28.41.6Inorganically grown carbonates[23]Aragonite (salinity 10 psu)−10.90.8Calcite (salinity 50 psu)−1.90.8Seawater29.70.4[28]BHVO-1 rock standard5.01.5Seawater 680 W30.70.4[29]Mediterranean Sea30.590.26[30]Red Sea30.490.12Dead Sea28.780.11Yarkon spring15.140.21SC -1 olivine3.41.0[31]Ia/211 clinopyroxene−2.41.0M 1 saprolite−11.61[32]10 saprolite0.21SH 65 Ocean Island basalt7.0<1[35]KRS9806 xenolith, Japan−7.70.83[36]9708 xenolith, Australia6.00.83Zagami meteorite4.40.5[38]15495 lunar low-Ti mare basalt5.60.214-1 amphibolite0.91[39]12-2 amphibolite−14.21
Bryant et al. [29] studied Li isotope ratio measurements by MC-ICP-MS under “cold plasma” conditions. The results are characterized by fewer baseline interferences and improved reproducibility as compared with conventional hot plasma techniques. The 2σ precisions for 1,200-W, 800-W, and 680-W plasma energy are conservatively estimated as 1.1‰, 0.7‰, and 0.5‰, respectively. The effects of analyte, acid, and matrix concentrations were discussed.
Seitz et al. [31] determined the Li isotopic composition of coexisting olivine, clinopyroxene, and orthopyroxene from spinel- and garnet-bearing peridotite xenoliths. The degree of intramineral fractionation correlated negatively with equilibration temperature.
The lithium isotopic composition of saprolites developed on a granite and diabase dike from South Carolina was measured to document their behavior during continental weathering. A general trend of decreasing δ7Li with increasing weathering intensity was observed [32, 33]. A similar study was carried out by Kısakürek et al. [34]. The internal precision on 7Li/6Li measurements was usually less than 0.20‰, and the external precision of Nu Plasma MC-ICP-MS, was 0.8‰.
Ryan et al. [35] defined lithium isotope variations in mantle sources (volcanic rocks from the Antarctica oceanic islands), and Nishio et al. defined lithium isotope variations in mantle-derived xenoliths [36]. These isotopic measurements have provided much information on the mantle source region. Similarly, Elliott et al. [37] showed that Li isotopes promise to provide significant new constraints on the distribution of recycled material in the mantle and its implications for mantle convection.
Lithium isotope compositions and concentrations of lunar samples, including basalts, breccias, and glass, and martian meteorites were measured using Thermo Finnigan MC-ICP-MS [38].
Teng et al. [39] measured lithium isotopic compositions in the country-rock amphibolites and schists using Nu Plasma MC-ICP-MS. δ7Li decreased dramatically with contact distance (along a −10 m traverse from the pegmatite into amphibolite) from +7.6 to −19.9.
Pogge von Strandmann et al. [40] investigated the behavior of Li isotopes in glaciated basaltic terrain. The δ7Li value of the suspended load was always lower than that of the bedload due to preferential retention of 6Li in secondary minerals during weathering. In turn, the δ7Li value of the dissolved load was always greater than that of the bedload. The authors suggested that δ7Li decreases with increasing chemical weathering. Similarly, Hathorne and James [41] used Li isotopes in seawater as a tracer for silicate weathering and showed that between 16 and approximately 8 Ma silicate weathering rate has increased, while weathering intensity has decreased.
Nishio et al. [42] studied Li isotope ratios in North Atlantic and Indian Ocean waters and its relation to those of Sr and Nd. The Li isotope results support the recent proposal that significant amounts of recycled lower continental crust might produce the radiogenic isotope signatures of the Indian Ocean basalts. Rudnick and Ionov [43] examined Li isotopic disequilibrium in olivine and clinopyroxenes from far-east Russia. Jeffcoate et al. [44] studied Li isotope fractionation in peridotites and mafic melts. Their results highlight the potential of Li isotopes as a high resolution geospeedometer of the final phases of magmatic activity and cooling. Wunder et al. [45] studied Li isotope fractionation between Li-bearing staurolite, Li-mica, and aqueous fluids.
Magnesium
Magnesium has three naturally occurring isotopes at mass numbers 24, 25, and 26 with relative abundance of 78.99, 10.00, and 11.01%, respectively. Galy et al. [46] briefly summarized the various fields where natural variations in the isotopic composition of this element may arise: (1) stellar nucleosynthesis and incorporation of presolar grains into meteorites, (2) the decay of 26Al to 26Mg, (3) isotopic fractionation in volatilization/condensation reactions, (4) isotopic fractionation during low temperature fluid/rock interactions, and (5) kinetic and thermodynamic isotope effects in biological processes.
Galy et al. [46] performed high precision magnesium isotope ratio measurements using MC-ICP-MS for three groups of materials: commercial Mg materials including the NIST isotopic standard SRM 980, natural magnesite, and commercial chlorophyll in spinach. The observed ratio 26Mg/24Mg was 0.139828 ± 0.000037 (2σ), compared to the NIST value of 0.13932 ± 0.000 6 (2σ) [47]. The variations in the Mg materials reported in δ25Mg and δ26Mg units are given in Table 2. A desolvation nebulizer was used to minimize the introduction of H2O, CO2, O2, and N2, therefore reducing the presence in the plasma of molecular interferences such as , C2H+, , CN+, and NaH+. Other possible interferences are doubly charged ions such as 48Ca2+, 48Ti2+, 50Ti2+, 50V2+, 50Cr2+, and 52Cr2+. These elements, if present, should be removed by purification.
Table 2Mg isotopic composition in standards and selected natural samples relative to DSM-3 (‰)Sampleδ26Mg±2σδ25Mg±2σReferenceAldrich Mg solution2.600.171.330.08[46]aDead Sea Mg metal3.960.152.030.08AG 177 magnesia2.030.041.030.01OUM 10988 magnesite, Italy1.220.040.600.03AG 27 chlorophill b, spinach1.060.070.540.03North Atlantic Sea water2.590.041.330.08[51]aMixed foraminifera−1.880.16−0.990.10Dolomite1.640.040.880.01Seawater[52]aEPR1 surface1.950.140.980.06WP45N 5800 m2.010.161.000.04Med-T1 surface2.020.071.050.09Hydrothermal 2,650 m−1.010.16−0.510.06River water MB160.760.460.380.22MB60.090.220.040.11PH60.220.060.110.06PH5−1.010.16−0.510.14Ganges−1.390.06−0.70.09[53]Amazon−1.030.07−0.530.04Lena−1.280.08−0.660.02Seawater−0.840.130.430.15[54]MT 66 solute−1.740.07−0.900.07Ett113 bulk silicate rock−0.420.03−0.210.02M 201 biotite−0.070.09−0.010.00MO33 100–110 soil0.020.010.010.01Ace78 travertine−4.010.11−2.060.07Dead Sea water−0.600.08−0.330.10[55]Seawater−0.740.05−0.360.12Sataf spring−2.500.10−1.300.10Carbonaceous chondrites[56]144A, Al-Ti diopside0.110.06−0.090.12144A, Al-Ti diopside−2.740.11−1.740.11Olivines[57]DR9894 Australia−1.430.2−0.660.13ZS56-2 Siberia−1.050.2−0.540.13aRelative to NIST isotopic standard SRM 980
Variations of the Mg isotope ratio in the metallic chips of the NIST SRM 980 magnesium isotopic standard were shown using five different MC-ICP-MS instruments of two types [48]. The chips were 1–50 mg in size. The differences in δ25Mg and δ26Mg of the SRM 980 were up to 4.2 and 8.19‰, respectively, while the long-term repeatability of the δ values was up to 0.09 and 0.16‰ respectively. Because of the heterogeneity of the NIST reference material, two homogeneous isotope standards, DSM-3 (Dead Sea magnesium) and Cambridge1 were prepared and characterized. The heterogeneity of Mg isotopes was also reported by Zhu et al. [49]. Carignan et al. [50] discussed the isotopic homogeneity of existing reference materials and suggested the acceptance of DMS-3 as a new Mg isotope reference standard material.
Chang et al. [51] developed a Mg separation technique for low-Mg biogenic carbonates with yields close to 100%. The technique was applied to the determination of Mg isotopes in three natural samples: seawater, foraminifera, and dolomite. De Villiers et al. [52] established the magnesium isotopic composition of seawater and evaluated its constancy as a function of depth and geographic location. The authors demonstrated that the magnesium isotopic composition of ancient oceans can be used to make important inferences about the relative contribution of different lithologies to the global continental weathering flux.
Tipper et al. [53, 54] analyzed river water, rock, travertine, and soil and demonstrated that both Ca and Mg isotope ratios are fractionated during weathering. The Mg isotope composition of the rivers is intermediate between limestone and silicate rock. Silicate soil has a δ26Mg of −0.03‰, heavier than that of silicate rock by 0.5‰. This fractionation in the soil creates a complementary groundwater reservoir of light Mg. Seasonal variations in Mg isotope ratios in the dissolved load are small, but define an array which can be modeled as a mixture between a fractionated groundwater reservoir and surface runoff.
Mg isotope fractionation during brine evolution in the Dead Sea is presently being studied using the MC-ICP-MS technique [55]. Samples from the solar system were used for 26Al−26Mg dating, by measuring the variations in the Mg ratios using laser ablation combined with MC-ICP-MS [56].
Pearson et al. [57] investigated Mg isotopic variations in the lithospheric mantle by analyzing olivine in mantle-derived peridotite xenoliths and megacrysts using a laser-ablation microprobe and MC-ICP-MS. δ26Mg ranges from −3.01 to +1.03‰ and δ25Mg from −1.59 to +0.51‰, relative to the magnesium isotopic standard DSM-3, were found. The in situ measurement of Mg isotopes thus provides a powerful new method for investigating processes in the mantle.
Calcium
Calcium has six stable isotopes at mass numbers 40, 42, 43, 44, 46, and 48 with relative abundance of 96.941, 0.647, 0.135, 2.086, 0.004, and 0.187%, respectively. Halicz et al. [58] studied in detail the MC-ICP-MS isotope ratio measurements of calcium. The more important points are discussed here. Under normal instrumental operation conditions it is not possible to use the isotope 40Ca because it is masked by the intense 40Ar+ ion beam. Furthermore, the ion dispersion of Ca isotopes is too large to allow the 48Ca−42Ca mass range to be accommodated on the multicollector array; therefore only 42Ca, 43Ca, and 44Ca are monitored. Ar-scattered interferences were monitored at half masses for elevated background correction. Corrections of doubly charged Sr were made by measuring doubly charged 87Sr2+. A desolvation nebulizer was used to reduce molecular interferences such as ,  , and . The results for the Ca ratios in the studied sample are given in δ units in Table 3 and were derived using the bracketing technique relative to the NIST SRM 915a Ca standard.
Table 3Ca isotopic composition in standards and selected natural samples relative to NIST SRM 915a (‰)Sampleδ44Ca±2σReferenceCaCO3 Merck0.610.28[58]2-8-E3 speleothem0.250.32Acropora coral0.580.16Shell from marine organism[59]USGS EN-10.53a0.07CaCO3 JM 9912−0.630.07CaCO3 JM 4064−6.620.08Inorganic calcite[61]CM0400.090.2CM039−0.070.2Growth solution0.310.2Foraminifera[62]OMEX-12b0.210.10WIND-10b0.280.11Mediterranean Sea water0.980.14Seawater1.090.09[54]MT 66 solute0.540.06Ett113 bulk silicate rock0.310.05MO33 0-10 soil0.510.05MO33 100-110 soil0.540.13Ace78 travertine0.140.05Carbonate fluorapatite[63]OG-50.260.11D4/010.420.08S30/030.230.0132a/010.520.1232/01s0.130.03D49/01−0.230.06aRelative to IAPSO seawater
Wieser et al. [59] developed a high precision Ca isotope ratio measurement technique for a Finnigan Neptune magnetic sector ICP-MS. Delta values including δ44Ca/43Ca, δ44Ca/42Ca, and δ48Ca/42Ca were measured with an external reproducibility better than 0.2‰ in seawater and biogenic and non-biogenic marine carbonates. Fietzke et al. [60] developed a new technique for direct measurements of 44Ca/40Ca ratios on an MC-ICP-MS using “cool plasma”. Reducing the plasma energy from the usually applied approximately 1,300 W to about 400 W significantly reduced the 40Ar+ isobaric effect, allowing simultaneous and precise measurements of the two Ca isotopes. Repeated measurements of the 44Ca/40Ca ratios in various Ca standard materials were in good agreement with data reported in the literature.
Marriott et al. [61] investigated temperature dependence of Ca isotopes in solution, inorganic calcite, and foraminifera and concluded that they are lighter than in the growth solution, and only weakly dependent on temperature. Sime et al. [62] also found negligible temperature dependence of calcium isotope fractionation in planktonic foraminifera.
River water, rock, travertine, and soil were studied by Tipper et al. [54] who demonstrated that Ca, as well as Mg isotope ratios, are fractionated during weathering. Fractionation of Ca during continental weathering is of importance to the global cycle of Ca. The riverine input of Ca to the oceans is controlled not only by the composition of the primary continental crust, but also by the size and composition of the fractionated reservoir on the continents. The impact on the oceanic cycle of Ca depends on the relative residence times of dissolved Ca in the ocean and the storage time of fractionated Ca.
Soudry et al. [63] explained fluctuations of Tethyan phosphogenesis through time, and whether or not they reflect long-term changes in ocean circulation or in continental weathering. A δ44Ca increase during the Late Cretaceous–Eocene also reflects a decrease in weathering Ca2+ fluxes together with increased biological removal of isotopically light Ca2+, fostered by increased continental flooding and concomitant carbonate (chalk) sedimentation on shelves. The overall concordant trends between the measured δ44Ca, the eustatic sea level curve, and the sizes of the flooded continental area throughout the Early Cretaceous–Eocene, point to a linkage between the Ca isotopic composition of paleoseawater and long-term paleogeographic and oceanographic changes.
Steuber and Buhl [64] analyzed the calcium isotope composition in modern and ancient marine carbonates. No significant temperature dependence of Ca isotope fractionation was found in Cretaceous shells. δ44Ca of Cretaceous seawater was 0.3–0.4‰ lower than that of the modern ocean.
Chu et al. [65] developed a procedure for the precise determination of Ca isotope ratios in natural and organic samples, such as bones, milk, and other biological materials. The data demonstrated that geological/environmental conditions do not cause large variability and it was suggested that diet is the major cause for variations in bones; so Ca isotope ratios may serve as a paleodiet tracer.
Skeletal carbonates from the Tethyan realm were analyzed by Farkaš et al. [66]. The observed late Mesozoic δ44Casw was simulated using a Ca isotope mass balance model, and the results indicated that the variation in δ44Casw can be explained by changes in oceanic input fluxes of Ca that were independent of the carbonate ion fluxes.
Sime et al. [67] interpreted Ca isotope behavior in marine biogenic carbonates. The 18 million year record of planktonic foraminifera in the Atlantic averages  and is a good match to Neogene Ca isotope record based on foraminifera, but is not similar to those in bulk carbonates. There are also publications on Ca using seawater as a standard for normalization in the bracketing method.
Copper
Copper isotope ratio measurements have been used to determine natural isotope variations in ore geology, geochronology, and archaeometry [68, 69]. Three different types of ICP-MS instrument, a quadrupole, a single collector magnetic sector, and a multicollector magnetic sector instrument, were compared by Diemer et al. [70]. Although precision of results significantly differed, excellent agreement was observed between results obtained using all the instruments. The results for the Cu-isotopic composition are given in Table 4.
Table 4Cu isotopic composition in standards and selected natural samples relative to SRM NIST 976 (‰)Sampleδ65Cu±2σReferenceNative copper[71]aOUM15126 Michigan, USA0.450.06OUM00061 Yekaterinburg, Russia−0.330.06OUM15127 Cornwall, England0.410.06Minerals OUM23585 malachite, England−0.260.06OUM1616 azurite, USA1.590.06OUM25139 chalcopyrite, England0.070.06Acc. Ser. 25300 chalcopyrite, Canada0.400.06G-4 chalcopyrite, sulfide deposits−0.440.06Ultra-pure Cu standards[75]JMC Cu Axiom0.6190.058JMC Cu IsoProbe0.6410.019IMP Cu IsoProbe0.200.10IMP Cu IsoProbe0.2070.049Copper sulfide precipitates[78]A-Cu- 0 Cu(I)S bulk0.700.06A-Cu- 0 Cu(I)S precipitated−2.520.06A-Cu-48 Cu(I)S bulk0.620.06A-Cu-48 Cu(I)S precipitated−2.320.06C-Cu-0 Cu(OH)2 bulk−0.260.06C-Cu-0 Cu(OH)2 precipitated−0.540.06Chalcopyrite from the Grasberg deposit region[73]aF1-001 Grasberg skarn0.1070.031XC05-001 Ertsberg diorite0.6330.035ES-005 Ertsberg skarn0.1930.054XC22001 pyrite shell0.2180.023XC25-002 bornite-2.69 Dalum−0.2690.031Alexandrinka VHMS sulfides[76]SW-1(a) chalcopyrite0.184<0.07SW-1(b) quartz0.318<0.07HV-1(a) sphalerite0.330<0.07CA-1 pyrite0.054<0.07CB-1(b) covellite−0.300<0.07CB-2(a) silicate−0.058<0.07Supergene copper sulfides[81]Tt-1, El Teniente bornite0.370.16Tt-6, El Teniente chalcopyrite−0.150.16El-1, El Salvador chalcopyrite0.810.16M-1, Mocha chalcopyrite−0.060.16aOriginal results were given in ɛ units (parts per 10,000) and have been converted to δ units
MC-ICP-MS ratio determinations of copper were comprehensively studied by Zhu et al. [71]. A sample purification procedure was described, noting that possible isotope fractionation must be avoided. Potential interferences in 65Cu/63Cu ratio measurements are (23Na40Ar)+ and  at mass 63 and (25Mg40Ar)+ at mass 65. It has been shown that for samples where Na/Cu and Mg/Cu ratios are below 10−4 and 10−3, respectively, and under instrumental working conditions applied in this study, the abundances of the polyatomic ions relative to Na+ and Mg+ were approximately 10−4, i.e., isobaric interferences were negligible.
Two Cu isotope measurement procedures were used: the standard-sample-standard bracketing technique where variations are calculated relative to Cu isotope standard (SRM NIST 976) as in Eq. 2, and the “doping” technique [2] where the sample is doped with Zn at a concentration Cu/Zn ratio close to 1. Early results were given in “ɛ” units, but in the last papers “δ” is accepted. A constant value of the 68Zn/66Zn ratio was used as a normalization factor to correct the measured 65Cu/63Cu ratios. External precision (2σ) better than 0.03‰ and 0.06‰ was achieved by applying the doping and bracketing techniques, respectively [71]. Borrok et al. [72] presented a new method for efficient separation of Cu, Fe, and Zn from the greater concentrations of matrix elements using a single anion-exchange column with hydrochloric acid media.
The possibility of laser ablation combined with a MC-ICP-MS for Cu isotope ratio determinations was presented by Graham et al. [73] About 160 solid samples were analyzed with a 2σ uncertainty of approximately 0.04‰. Jackson and Günter [74] examined the influence of various processes of laser ablation on fractionation of the Cu isotope ratio. The data suggest that the dominant source of isotopic fractionation at high laser fluence was the preferential volatilisation of 63Cu during incomplete vaporization and ionization in the ICP of particles greater than approximately 0.5 μm in diameter.
Mason et al. [5, 75] performed a detailed study on spectral interferences across the mass range 63Cu to 70Zn and the mass discrimination corrections using two different MC-ICP-MS instruments: the double focusing VG Axiom and the single focusing Micromass IsoProbe. It was observed that the ion types and their intensities depend on the different instrumental configuration. The importance of removing problematic matrix components prior to the Cu and Zn measurements was also emphasized. Based on their method Mason et al. [76] studied Zn and Cu isotopic variability in the Alexandrinka sulfide ore deposit from the Urals, Russia. A further study on mass discrimination correction shows the importance of matrix removal, particularly Fe and Ti, the dependence of mass discrimination on Cu/Zn ratio in the solution and that sample introduction system with a desolvating membrane causes variable behavior of the Cu standard, probably due to variations in Cu oxidation state in the solution [77].
Ehrlich et al. [78] studied copper isotope fractionation between aqueous Cu(II) and CuS, the latter being precipitated from CuSO4 solution with Na2S under anoxic conditions. Using Ni as a doping agent and the standard-sample-standard bracketing technique a 2σ error of 0.06‰ was achieved. The mean fractionation factor at 20 °C was derived: . Additional experiments over the temperature range 2–40 °C showed an inverse dependence of the fractionation factor on temperature. Markl et al. [79] used copper isotopes as monitors of redox processes in hydrothermal mineralization. The authors concluded that copper isotope analyses cannot be easily used as a reliable fingerprint for the source of copper in archaeology and geology because the variation caused by redox processes within a single deposit is usually much larger than between deposits. However, Asael et al. [80] showed that systematically large Cu isotope fractionation occurred during redox processes in sedimentary copper ore deposits. Cu isotope fractionation was also observed in bacterial oxidizing environments [81].
The roles of Cu and Zn isotopes in chondrites and iron meteorites were studied by Luck at al. [82, 83]. A typical error (external precision) for δ65Cu ± 0.4–0.5‰ was reported. Moynier et al. [84] investigated isotopic composition of zinc, copper, and iron in lunar samples.
Recently isotopic ratio studies of Cu and Zn were performed in seawater [85]. The very low concentration of these elements and high TDS matrix demanded development of preconcentration and purification, followed by separation processes. In this work accurate data and their uncertainties are given only for Zn.
Conclusions
We have reviewed a group of more than 60 papers that were published in the period from 2000 up to present. We also quote results from a few unpublished works made at the GSI. We have chosen to focus on the isotope ratio determinations of only four elements, namely lithium, magnesium, calcium, and copper, where high precision ratio measurements were for various reasons impossible to achieve with the early MC-ICP-MS instruments. The second-generation machines have detection systems with sufficient dispersion to accommodate Li ions and the capability to be tuned to partial mass numbers. The high resolution capability allows one in some cases to resolve interferences. New types of desolvation nebulizers allow significant reduction of solvent and atmospheric interferences and the “cold plasma” technique reduces nebulizing gas ion intensities. Consequently, precision (±2σ) for δ7Li, δ25, 26Mg, δ44Ca, and δ65Cu down to the range of (0.1–0.2)‰, (0.01–0.1)‰, (0.05–0.1)‰ and (0.06–0.15)‰, respectively, can now be achieved.


MRI evaluation of tissue iron burden in patients with β-thalassaemia major

β-Thalassaemia major is a hereditary haemolytic anaemia that is treated with multiple blood transfusions. A major complication of this treatment is iron overload, which leads to cell death and organ dysfunction. Chelation therapy, used for iron elimination, requires effective monitoring of the body burden of iron, for which serum ferritin levels and liver iron content measured in liver biopsies are used as markers, but are not reliable. MRI based on iron-induced T2 relaxation enhancement can be used for the evaluation of tissue siderosis. Various MR protocols using signal intensity ratio and mainstream relaxometry methods have been used, sometimes with discrepant results. Relaxometry methods using multiple echoes achieve better sampling of the time domain in which relaxation mechanisms take place and lead to more precise results. In several studies the MRI parameters of liver siderosis have failed to correlate with those of other affected organs, underlining the necessity for MRI iron evaluation in individual organs. Most studies have included children in the evaluated population, but MRI data on very young children are lacking. Wider application of relaxometry methods is indicated, with the establishment of universally accepted MRI protocols, and further studies, including young children, are needed.
Introduction
β-Thalassaemia major is a hereditary anaemia characterized by ineffective erythropoiesis and haemolysis [1]. The underlying mechanism is defective production of haemoglobin β-chains, resulting in excess of α-chains, which are unstable and precipitate to form intracellular inclusion bodies [2, 3]. This excessive intracellular deposition of α-chain material is responsible for accelerated apoptosis of the erythroid precursors and for peripheral haemolysis of the erythrocytes [3]. By the age of 3 months, severe anaemia develops leading to increased intestinal iron absorption. To maintain haemoglobin at a level of 10–12 g/dl, patients suffering from β-thalassaemia major need to be given repeated blood transfusions [1]. A major drawback of this treatment is transfusion siderosis, which, in association with the increased intestinal iron absorption, apoptosis of the erythroid precursors and peripheral haemolysis, leads to iron overload [1].
Iron is ubiquitous in eukaryotic organisms. It is essential for cellular survival and proliferation and for haemoglobin synthesis [4, 5]. Human demands for iron are covered partly by intestinal absorption, but mainly from the recycling of iron from old or abnormal erythrocytes phagocytized by macrophages [6]. Iron released from macrophages binds mainly to transferrin, but also to citrate and albumin to form non-transferrin-bound iron (NTBI) which is a toxic form [7–9]. Extracellular iron bound to transferrin enters the cell via transferrin receptors by a process of endocytosis. Once iron is released into the cytoplasm, it enters a poorly defined cellular compartment termed the labile iron pool (LIP). Iron in this compartment is loosely bound and therefore is highly toxic [10]. Intracellular iron that is not needed for immediate use is stored in the form of ferritin which consists of an apoprotein storing up to 4,500 atoms of iron (loading factor 4,500) [5, 11], and is not cytotoxic. When ferritin storage capacity is exceeded the LIP increases and haemosiderin is generated from ferritin denaturation [10]. Iron in the form of haemosiderin is thought to be more cytotoxic [10]. The iron in the LIP is in both the ferrous (Fe2+) and ferric (Fe3+) forms [12]. Fe2+ reacts with hydrogen and lipid peroxides and generates highly toxic hydroxyl and lipid radicals (Fenton reaction) that damage cellular membranes, proteins and nucleic acids [4]. Iron overload, just as lack of cellular iron, may lead to cell death and organ dysfunction.
Iron overload is a major cause of morbidity and mortality in β-thalassaemia major [1]. Iron accumulates initially in the reticuloendothelial system (bone marrow, spleen, liver) and then in the hepatocytes, the heart (myocytes) and the endocrine glands [1, 9, 13]. In contrast to the reticuloendothelial cells, the turnover of iron in the hepatocytes, myocytes and endocrine glands is very low [9]. Chelation therapy has been used to eliminate excess iron [1, 7]. Chelatable iron is derived from the catabolism of haemoglobin in macrophages [14]. Chelators remove NTBI from the plasma, but they do not interact with ferritin and haemosiderin at clinically relevant rates [10, 15]. Desferrioxamine (DFO), which is the chelating agent most widely used over the last 30 years, requires parenteral administration. It removes mainly extracellular iron and only a fraction of the intracellular LIP iron [14, 15]. Deferiprone is an oral iron chelator that penetrates the cellular membrane and chelates intracellular species of toxic iron [16]. Cellular uptake of chelators takes place at different rates in different cells [15]. DFO is of low toxicity, mainly affecting the optic, auditory and skeletal systems [7]. Skeletal changes develop mainly due to the toxic effects of DFO on the growth cartilage and are manifested as disproportionate truncal shortening with loss of seated height [7]. Adverse effects of deferiprone are arthralgia, gastrointestinal symptoms, elevated liver enzymes and rarely agranulocytosis [7, 16].
The effective management of patients, and especially of children, with thalassaemia requires optimal monitoring of the toxic effects of both iron overload and excessive chelation therapy. Serum ferritin has been widely used as a surrogate marker and a target ferritin level of 1,000 μg/l is generally recommended [7, 17]. However, serum ferritin represents only 1% of the total iron pool, and as an acute-phase protein it is not specific because the levels can be raised in inflammation (e.g. hepatitis) and liver damage [7, 18, 19]. Chronic liver inflammation is not rare in patients with thalassaemia, since over 40% of them have positive anti-hepatitis C virus (HCV) antibodies and more than 50% have chronic (persistent or active) hepatitis [20, 21]. Liver iron concentration (LIC) measured on needle biopsy is currently considered the gold standard for the evaluation of siderosis [7, 18, 19]. However, needle biopsy is an invasive technique, it is not easily repeatable and the accuracy of the resulting LIC measurement is greatly affected by hepatic inflammation-fibrosis and uneven iron distribution [22]. Furthermore, it appears that cardiac iron overload, which is the leading cause of death in thalassaemia, cannot be predicted from the degree of liver siderosis, probably because of differences in iron kinetics between liver and heart cells [9]. The use of non-invasive techniques for monitoring iron overload in each of the affected organs would be preferable, and to this end MRI has been used increasingly over the last two decades [19, 22–44]. Other non-invasive methodologies are too complex, too costly and not readily available (i.e. magnetic susceptometry), or lack imaging capability (i.e. magnetic resonance spectroscopy) [45, 46].
MRI assessment of tissue iron
Relaxation theories
MRI evaluation of tissue iron overload is based on T2 relaxation enhancement induced from the interactions between high-molecular-weight iron complexes such as ferritin, haemosiderin and ferrioxamine with water molecules [5, 47, 48]. Ferrioxamine is produced after cellular uptake of DFO, and in extrahepatic tissues, where there is no active excretion and it may remain for days [7]. Ferrioxamine-based agents have been used in the past as contrast media because of their T2* effect [49]. “Inner sphere” and “outer sphere” relaxation theories have been proposed to explain the T2 relaxation enhancement induced in siderotic tissues [50–55]. According to the inner sphere theory, iron electron spins enhance the relaxation of protons of water molecules buried in iron-containing proteins. Enhanced relaxation of the bound water protons is then transferred to the free water protons by water or proton exchange [48, 56, 57]. According to the outer sphere theory, magnetic field gradients induced at the periphery of iron-containing proteins create loss of phase and relaxation enhancement of free water protons diffusing in the neighbourhood [53, 54].
MR methodologies
MR methods for assessing tissue iron can be separated into two groups: signal intensity ratio (SIR) methods and relaxometry methods. Various techniques have been described, including: (a) methods measuring SIR based on T2-weighted (spin-echo) or T2*-weighted (gradient-echo) sequences [19, 23–26, 30, 58–62], (b) relaxometry methods measuring absolute T2, (c) relaxometry methods measuring absolute T2*, and (d) hybrid relaxometry methods [30–32, 34–37, 39–43, 58, 63].
SIR methods
These have been used for the study not only of the liver but also of other organs such as the spleen, pancreas, pituitary gland, bone marrow and abdominal lymph nodes [19, 23, 25, 27, 30, 58–62]. For SIR assessment the signal intensity of the target organ is divided by the signal intensity of a reference tissue (e.g. fat, muscle) or noise. Signal intensity measurements are performed in the same slice by using the region-of-interest (ROI) method. For large organs such the liver, spleen and pancreas more than one ROI is used, positioned in areas lacking vascular structures and movement artefacts [19, 23, 25, 27, 30, 58, 61, 62]. The mean signal intensity from the different ROIs is then divided by the signal intensity of the reference tissue. One ROI is used for signal intensity measurement of the pituitary gland, bone marrow, abdominal lymph nodes and the reference tissue [58–60]. For SIR evaluation of the liver various reference tissues have been used, of which paraspinal muscle appears to be the best choice because it combines good sensitivity with minimum intersite variability [19, 64]. A disadvantage of the SIR methods is that in most cases they use only one echo time (TE) and thus lose their detection sensitivity in tissues with heavy siderosis, where transverse relaxation is much faster than the TE. This occurs particularly in the liver at the upper range of LIC values where signal intensities are widely dispersed [39]. Gandon et al. [19], by using an algorithm that combined signal intensity ratios from multiple sequences with different TEs, achieved extension of the detection range up to about 21 mg Fe per gram dry liver tissue, with sensitivity and specificity similar to those of biochemical analysis.
T2 relaxometry methods
These assess T2 relaxation time or R2 (1/T2) by using the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo sequence, which employs multiple (2–32) equidistant refocusing 180° pulses, each followed by an echo [65, 66]. Most scanners, by using a pixel-by-pixel, log-linear fitting model, automatically derive the corresponding T2 maps. Signal intensity measurements in the T2 maps correspond to the mean T2 relaxation time of the included voxels [67]. To avoid motion artefacts from respiration, studies of abdominal organs should be performed with respiratory triggering. To this end a pressure-sensitive pad is attached to the abdomen and depending on the patient’s respiratory cycle a delay time is chosen. Data acquisition takes place at the end-expiration phase [39, 40]. During cardiac MRI, in addition to respiratory triggering, cardiac triggering is necessary. To obtain a minimum repetition time (TR) of 2,000 ms two to three R–R intervals should be allowed between successive excitations [39, 40]. The mid-systole phase and a cardiac trigger delay of around 250–300 ms are usually chosen to image the myocardium with sufficient thickness for the best signal intensity measurements [39, 40]. A pseudo four-chamber or short-axis view of the heart is generally used. A T2 relaxometry method that received FDA approval for clinical liver iron estimation has recently been developed by St. Pierre et al. [44]. This method uses multiple T2-weighted single spin-echo sequences with different TEs acquired in half-Fourier mode to reduce acquisition time. The calculated mean R2 values combined with LIC values, obtained from liver biopsies, are used to create calibration curves.
T2* relaxometry methods
These evaluate T2* or R2* (1/T2*) by using multiple gradient-echo sequences with different TEs. These methods have been developed to further accelerate acquisition, in order to increase sensitivity and eliminate artefacts related to respiration or cardiac motion. To obtain R2* (1/T2*) values, the signal decay curve is usually fitted with an exponential model: S=S0e−TE/T2*, where S is the net image signal intensity, TE is the echo time and S0 is a constant [68]. T2* relaxometry methods have been used mainly for myocardial iron assessment and cardiac gating is always applied [35, 42, 68–71]. Breath-hold sequences have greatly eliminated motion artefacts [35, 42, 68].
Hybrid relaxometry methods
Hybrid approaches have been applied in high fields and measure both R2 and R2* to calculate the inhomogeneity factor R2′=R2*−R2 [34, 72, 73]. These approaches assume that R2′ is more specific to mechanisms of relaxation related to iron than R2 [34, 73].
Comparison of the MRI methodologies
SIR versus relaxometry
SIR methods require shorter acquisition times but lack a wide range of iron assessment [63]. Relaxometry methods, mainly the T2* method, by using multiple echoes create in- and out-of-phase effects between water and fat transverse magnetization (Fig. 1) [35]. Relaxometry methods, although taking longer, are preferable because they achieve a better sampling of the time domain in which relaxation mechanisms take place and lead to more precise results [63].
Fig. 1A 15-year-old male with β-thalassaemia major. Axial scan with a multiecho gradient-echo sequence (TR/TE 150/1.07, 2.14, 3.21, 4.28, 5.35, 6.42, 7.49, 8.56, 9.63, 10.7, 11.77, 12.84, 13.91, 14.98, 16.05, 17.12, 18.19, 19.26, 20.33, 21.4 ms; flip angle 35°). a Second echo shows pixel annulation (arrows) due to out-of-phase phenomena, at the interfaces of abdominal organs and muscles with fat. b Fourth echo: no pixel annulation is observed because water and fat protons are in phase (courtesy of Dr. M. Douskou)
T2, T2* and hybrid methods
There is no general consensus on which relaxometry method (spin-echo or gradient-echo) or index (R2, R2*, R2′) is best for tissue iron quantification. Theoretically, R2* and R2′ are more sensitive than R2 to iron-induced field inhomogeneities. Gradient-echo sequences evaluating R2* are preferred for cardiac relaxometry, because of their short acquisition time, but gradient-echo sequences are more prone to artefacts and R2* is more dependent on factors unrelated to iron, such as susceptibility artefacts from the lungs and the blood oxygenation level-dependent (BOLD) effect [35, 74]. According to the BOLD effect, the T2* decreases as the concentration of intracapillary deoxyhaemoglobin increases [74]. A pronounced BOLD effect may be observed in early diastole and results in signal loss [75, 76]. Data collection during mid-systole helps to overcome BOLD effect interference with accurate T2* measurements of the myocardium. In liver and pituitary studies both T2 and T2* have been assessed. T2* is more sensitive to low iron content, but studies in the liver show that these methods suffer from inaccuracies at high iron concentrations [77]. Furthermore, in high fields susceptibility artefacts from the sphenoid sinus are more pronounced with gradient-echo sequences and may lead to inaccurate measurements of pituitary siderosis [43].
MRI studies of individual iron overloaded organs
The degree of siderosis, the crystalline structure of ferritin, the rate of iron elimination under chelation therapy and the degree of ferrioxamine formation are all organ-specific [7, 15, 18, 78]. All these parameters may be responsible for differences in the T2 relaxation enhancement induced in the various organs. Individual organs should be considered separately, and the effect of age on iron overload should be taken into account. Higher survival probabilities have been reported in patients with thalassaemia born in the last 30 years [79]. Patient compliance with treatment regimens and effective chelation therapy are thought to be the main factors associated with improved survival [79]. The combination of DFO, deferiprone and the new oral chelators is considered very promising, but will require effective monitoring by non-invasive methods [80]. An increasing number of studies have evaluated iron in the various affected organs by MRI [19, 22–44]. Many of these studies have included paediatric patients, but very few have investigated children younger than 10 years and none has included children younger than 5 years [17, 18, 28, 39, 43, 58, 60–62, 77, 81–84]. In addition, very few studies have evaluated the effect of age on individual organ siderosis and only one has evaluated and compared MRI data from younger and older patients with thalassaemia [28, 81, 82, 84]. Studies have tended to focus on specific organs.
Liver
For the MRI evaluation of liver siderosis both SIR and relaxometry techniques have been used (Fig. 2) [18, 23, 28, 32, 44, 68, 85]. R2 of the liver demonstrates a significant positive correlation with serum ferritin and LIC determined from liver biopsy material [18, 23, 32, 39, 40, 44, 68]. Comparative evaluation of hepatic R2 and R2* in iron-overloaded patients demonstrates that both parameters correlate closely with LIC [85]. The relationship of SIR with LIC and serum ferritin varies among studies [18, 30, 58]. In most studies R2 and SIR show a better correlation with LIC than with serum ferritin [18, 23, 28, 32, 44, 68, 85]. This can be explained in part because the HCV-positive thalassaemia patients in the studies had higher serum ferritin levels than those who were HCV-negative [77, 86]. Liver R2 shows no association with the hepatic inflammation histological activity index or the type of hepatitis (chronic persistent or chronic active), but is affected by hepatic fibrosis [18, 39, 77].
Fig. 2A 17-year-old male with β-thalassaemia major. Axial scan, fourth echo of a multiecho spin-echo sequence (TR/TE: 2000/20, 40, 60, 80, 100, 120, 140, 160 ms), shows low-signal intensity in the liver, bone marrow of the spinal body and the pancreas suggesting iron overload
In iron overload states, over 70% of body iron is found in the liver and LIC has been considered to be the best marker of total body iron burden [7, 18, 19]. Based on the good correlation between hepatic R2 or SIR and LIC a number of recent studies have tested the relationship between siderosis of the liver and other organs [28, 39, 40, 58–61, 87]. No correlation has been found between liver and pituitary siderosis [28, 58]. With regard to the heart, a correlation with liver siderosis has been found only in cases of heavy myocardial iron deposition [39, 40]. This lack of correlation can probably be explained by differences in transferrin receptor concentration, iron kinetics, the crystalline structure of ferritin and the degree of organ inflammation or fibrosis [7, 78, 88–92]. Furthermore, under chelation therapy with DFO intracellular paramagnetic ferrioxamine is formed, which exits slowly from cells unless there is an active excretion pathway as is present in the hepatocytes [88].
Young patients with thalassaemia studied longitudinally have shown absence of substantial improvement in the MR parameters of liver siderosis under different chelation therapy regimens [82]. This may be explained by the fact that liver siderosis progresses very fast in thalassaemia patients, and iron overload develops after only 2 years of transfusion therapy [93]. Therapy with the most widely used chelating agent is started at the age of about 3 years, and until growth is completed DFO should not exceed a dose of 40 mg/kg per day [7]. An early start to monitoring the progress of tissue iron deposition with MRI might be useful in deciding whether to begin chelation therapy at a younger age and when to introduce new chelating agents.
Heart
Cardiac failure is the leading cause of death from iron overload in patients with thalassaemia [94, 95]. Thalassaemia patients on a regimen of regular blood transfusions who are not receiving chelation therapy, develop heart enlargement by the age of 10 years and heart failure by the age of 16 years [22]. Cardiomyopathy secondary to iron overload is potentially reversible with vigorous chelation therapy [7]. Myocardial iron has been evaluated by MRI using SIR and relaxometry techniques (Fig. 3) [22, 28, 39, 40, 68, 82, 96–98]. In the prechelation era, post-mortem examination of patients with thalassaemia showed a close correlation between cardiac and hepatic iron concentrations. MRI studies have demonstrated discordant results regarding the relationship of myocardial iron with hepatic iron and serum ferritin [28, 39, 40, 68, 97]. Differences in iron kinetics and variations in chelation schemes may be responsible for the lack of correlation of the MRI-determined myocardial iron with that of the liver [88]. More active elimination of iron from the hepatocytes than from the myocytes may play a role in the absence of correlation in patients receiving chelation therapy [28, 39, 68].
Fig. 3A 16-year-old male with β-thalassaemia major. a T2 map of the short axis of the heart shows low values of the left ventricle (arrow) and the septum (arrowhead). b R2* (=1/T2*) map of the short axis of the heart shows high signal of the septum (asterisk). Note the reduction of motion artefacts and better delineation of the heart boundary in the R2* image (courtesy of Dr. M. Douskou)
Another important issue is whether myocardial iron evaluated by MRI could predict which patient will develop cardiac failure and arrhythmia. Thalassaemia patients with significantly reduced left ventricular ejection fraction (LVEF) have myocardial T2* values about 20 ms [22, 37, 38, 68], although most patients with T2* values less than 20 ms have normal LVEF [68]. Furthermore, the relationship between myocardial R2 and LVEF is either non-existent or weak [22, 38–40]. Differences in cardiac functional indices among patients with similar MRI measurement of iron might also be explained by genetic factors that predispose to the more rapid development of cardiac failure when a critical level of myocardial siderosis is reached [99, 100].
Heterogeneous iron distribution in the myocardium has been demonstrated in histological studies, and this would suggest the necessity for global MRI estimation of myocardial iron [42, 101]. Pepe et al. [42], using T2* multiecho cardiac MRI, demonstrated that global T2* shows a close correlation with midseptal T2*, suggesting that the latter may be used for a quick assessment of myocardial siderosis.
MR studies have shown that cardiac siderosis increases with age, but that siderosis progresses more slowly in the heart than in the liver [28, 82]. Systematic MRI studies in children are necessary in order to evaluate the age at the start of cardiac iron overload.
Pituitary gland and brain
Increased iron deposition in the anterior pituitary gland is the cause of hypogonadotropic hypogonadism and growth hormone deficiency [102, 103]. Iron accumulates in all five cell types of the adenohypophysis, but preferentially in the gonadotropin-secreting cells [102, 103]. Gonadotropin cell death due to iron toxicity is probably the cause of the decreased pituitary gland height observed in thalassaemia patients with hypogonadotropic hypogonadism (Fig. 4) [104]. MRI has been used to evaluate pituitary siderosis with SIR and relaxometry techniques [28, 43, 83, 105]. SIR and R2 findings show significant correlation with serum ferritin [83]. The very few studies evaluating simultaneously the pituitary and other solid organs have demonstrated no correlation between their MRI parameters of siderosis [28, 43, 81]. Pituitary siderosis appears to increase with age, but MR data are lacking for children under the age of 7 years [28, 81] [84].
Fig. 4The pituitary. a A 12-year-old male with β-thalassaemia major. Midsagittal T1-weighted (TR/TE 500/20 ms) scan shows low-signal intensity of the anterior pituitary lobe (arrow) and the bone marrow (asterisks) suggesting iron overload. The pituitary gland is small measuring 3.5 mm (normal for age 5.3±0.8 mm) [112]. This patient developed hypogonadotropic hypogonadism. b An 11-year-old male with β-thalassaemia major. Mid-sagittal T1-weighted (TR/TE 500/20 ms) scan shows normal signal intensity of the anterior pituitary lobe (arrow) along with normal pituitary gland height (6 mm). The bone marrow (asterisk) shows low-signal intensity suggesting iron overload
The single study assessing iron in the brain of thalassaemia patients by MRI [106] showed significantly higher R2 values in the cortex, the putamen and the caudate nucleus in patients than in controls. A lack of correlation between R2 and serum ferritin was found, possibly because serum ferritin, which does not cross the blood–brain barrier, is not a good marker of brain siderosis [106].
Pancreas
Iron overload leads to impairment of the endocrine and exocrine functions of the pancreas [107, 108]. Pancreatic siderosis has been evaluated in thalassaemia patients using SIR techniques and relaxometry techniques [61, 84, 109]. Midiri et al. [109], using fat as the reference tissue, found a significant negative correlation of SIR with serum ferritin and trypsin. Papakonstantinou et al. [61], using muscle as the reference tissue, found no relationship of the pancreatic SIR with the hepatic SIR or serum ferritin. The findings of Argyropoulou et al. [84], using T2 relaxometry techniques, were similar. In addition, relaxometry techniques demonstrated that the T2 relaxation time of the pancreas of patients with thalassaemia is significantly lower in children than in adults [84]. Fatty degeneration of the pancreas in adult patients may explain these differences (Fig. 5).
Fig. 5Siderosis of the spleen, pancreas and adrenals. a A 14-year-old male with β-thalassaemia major. Axial scan, fourth echo of a multiecho sequence (TR/TE 2,000/20, 40, 60, 80, 100, 120, 140, 160 ms) shows low-signal intensity of the liver, pancreas (arrow) and spleen suggesting increased iron deposition. b A 27-year-old-male with β-thalassaemia major and diabetes. Axial T2-W (TR/TE 1,800/80 ms) scan shows low-signal intensity of the liver and adrenals suggesting siderosis. High signal intensity of the pancreas (arrow) suggests tissue damage and fatty degeneration
Adrenals
Abnormalities in adrenal function have been reported in patients with thalassaemia [103]. There is only one study evaluating adrenals for iron overload with MRI (Fig. 5), which showed a significant correlation between adrenal and liver siderosis [87].
Spleen, lymph nodes and bone marrow
In spite of the fact that the spleen, lymph nodes and bone marrow, which all contain reticuloendothelial cells, are among the first organs to be affected by iron overload [1, 13], there have been very few studies evaluating their iron overload in thalassaemia by MRI, and these used mainly SIR techniques [59, 60, 62, 84]. SIR of the spleen shows a significant correlation with serum ferritin but not with SIR of the liver (Fig. 5) [62]. The absence of correlation between liver and spleen siderosis could be explained by differences in iron kinetics, by differences in the cluster size of iron proteins, by haemochromatosis gene mutations in β-thalassaemia major carriers, and by the presence of extramedullary haemopoietic tissue in the spleen [78, 110, 111]. Intraabdominal lymph nodes in β-thalassaemia have been related to chronic hepatitis C [60]. Lymph node siderosis correlates with liver, but not with spleen siderosis [60].
In the few studies that have been reported, SIR and relaxometry methods have shown discordant results for MR parameters of bone marrow siderosis and serum ferritin [59, 84]. Normal bone marrow signal associated with liver siderosis has been reported in a few patients with thalassaemia and this may be due to differences in genotype or differences in chelation therapy regimens [59].
Conclusion
It is evident that MR relaxometry has the potential to become the method of choice for non-invasive, safe and accurate assessment of iron load. Further theoretical research, along with studies monitoring wider age groups of patients are needed before a generally accepted protocol can be established. Until then, extreme caution is needed in its clinical application and interpretation. Experimental artefacts, non-optimized protocols, poor data analysis and unawareness of the inherent limitations of current methodologies in assessing a heavy body iron burden can result in misleading diagnosis and inappropriate management of thalassaemia patients with iron overload. Future experimental developments in relaxometry based on a better theoretical understanding of the contribution of the iron-containing proteins to the MR signal are expected to further strengthen its clinical role in the monitoring of patients with β-thalassaemia major.


Oxidative Stress and the Induction of Cyclooxygenase Enzymes and Apoptosis in the Murine Placenta

Placental oxidative stress has been implicated in many complications of human pregnancy, including preterm delivery and preeclampsia. It is now appreciated that reactive oxygen species can induce a spectrum of changes, ranging from homeostatic induction of enzymes to apoptotic cell death. Little is known regarding the occurrence of placental oxidative stress in other species. We investigated markers of oxidative stress in the labyrinthine (LZ) and junctional (JZ) zones of the murine placenta across gestational age, and correlated these with expression of the cyclooxygenase enzymes COX-1 and COX-2, and apoptosis. We tested a causal link between the two by subjecting placental explants to hypoxia-reoxygenation (H/R) in vitro, a known stimulus for generation of oxidative stress. Western blotting demonstrated significant increases in the concentrations of hydroxynonenal (HNE), COX-1 and COX-2 with gestational age. Dual-labelling demonstrated co-localisation of HNE, and COX-1 and COX-2 within the trophoblast of the LZ, and glycogen cells of the JZ. An apoptotic index based on TUNEL-positivity demonstrated an increase with gestational age, and dual-labelling showed co-localisation of TUNEL labelling with HNE and active caspase-3 within the trophoblast of the LZ. H/R significantly increased oxidative stress, induction of COX-1 and COX-2, and the apoptotic index. Co-localisation demonstrated the increases in COX to be within the trophoblast of the LZ, and in particular the glycogen cells of the JZ. Apoptosis was restricted to the LZ. We speculate that the induction of COX enzymes is a physiological response to oxidative stress, and may play a role in initiating or augmenting parturition. Generation of oxidative stress may also play a role in influencing the growth trajectory of the placenta, and its component cell types. The mouse may provide an experimental genetic model in which to investigate these phenomena.
1
Introduction
Oxidative stress of the placenta has been implicated in the pathogenesis of many complications of human pregnancy, including miscarriage, preeclampsia, and preterm labour [1–3]. These complications all share the common predisposing feature of reduced trophoblast invasion and incomplete conversion of the uterine spiral arteries [4,5]. Therefore, it is generally held that malperfusion of the placenta leads to increased oxidative stress, resulting in placental dysfunction. In the case of preeclampsia it is thought that the stress induces the release of a cocktail of factors, including pro-inflammatory cytokines, anti-angiogenic factors and apoptotic debris [6], into the maternal circulation that causes activation of the peripheral endothelial cells. Placental inflammatory lesions have also been associated with premature onset of labour [7].
By contrast, there are few data relating to placental oxidative stress available for other species. The mouse is potentially a powerful model in which to study the mechanisms of placental and obstetrical pathologies, and is the best-studied mammalian experimental genetic model system. The definitive murine placenta is a discoid haemochorial organ as in the human, and there are three anatomically and physiologically distinct regions: the labyrinth zone (LZ), junctional zone (JZ) and decidua basalis (DB) [8]. The labyrinth zone is the principal area of exchange, and consists of a meshwork of maternal blood spaces lined by trophoblast. The trophoblast comprises three layers; an outer layer (I) composed of cytotrophoblast cells, and two layers (II & III) of syncytiotrophoblast [9]. Deep to the syncytiotrophoblast are the fetal capillaries, embedded in a small amount of connective tissue. The junctional zone, interposed between the labyrinth and the decidua basalis, is of unknown function, but contains spongiotrophoblast cells and trophoblastic glycogen cells (GC) [8].
The decidua basalis is composed principally of maternal uterine tissues, and contains maternal arteries and veins that are continuous with the arterial and venous channels traversing the JZ. Glycogen cells migrate into the DB and surround the maternal arteries that supply the placenta late in gestation. The mechanisms of myometrial stimulation and activation appear to parallel those of the human [10]. For example, prostaglandins (PGs) are central components of labour in both humans and mice [11]. There are fundamental similarities in the changes occurring prior to labour, including an increase in myometrial contractile activity, increased coupling of myometrial cells through the formation of gap junctions, an increase in PG receptors in the myometrium, and enhanced sensitivity to oxytocin at term. There is also a rise in intracellular calcium resulting in an increase in myosin light chain phosphorylation.
In this study we determined the degree of oxidative stress present in the murine placenta at different gestational ages in normal pregnancies. We also tested whether there was a temporal and spatial association between oxidative stress and two markers of placental function, one physiological and one pathological. These were the induction of cyclooxygenase (COX) enzymes and apoptosis respectively.
Evidence from other systems points to potential links between oxidative stress and COX induction, resulting in the production of PGs [12–16]. PGs are derived from arachidonic acid (AA) through the actions of the COX enzymes, which are rate-limiting. There are two known isoforms of COX, COX-1 and COX-2, which share similar structures but differ in their function. COX-1 is believed to play a constitutive “housekeeping” role, whereas COX-2 is considered to be an inducible isoform. However, these contrasting roles have not been proven in the mouse [17]. In mice induction of COX-2 by administration of lipopolysaccharide leads to preterm labour, which can be blocked using the specific COX-2 inhibitor SC-236 [18].
Apoptosis has been described within the LZ of the normal mouse placenta, and is most frequent towards term and in post-mature placentas [19]. The stimulus for this is not known, but oxidative stress is a potent inducer of trophoblast apoptosis in the human placenta [20].
2
Materials and methods
2.1
Tissue collection
Placentas were collected from C57BL/6J inbred mice, and all experiments were carried out in accordance with the UK Government Home Office licensing procedures. Stages E14, E16, E18, and E19 were studied, where E1 of gestation was the morning when a copulation plug was found.
2.2
Western blotting
Placentas from three different animals at each gestational age were homogenized in ice-cold sample buffer, and centrifuged at 15,000 rpm. Protein concentration within the supernatant was determined using a colorimetric assay (Bio-Rad Bradford protein assay). An equal amount of protein from each sample (30 μg) was subjected to electrophoresis in a 10% SDS-polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membrane by a semidry transfer machine. Membranes were incubated in a blocking solution (TBS containing 5% powdered non-fat milk and 0.01% Tween-20) at room temperature for 1 h, and then in the same solution containing primary antibody (Table 1) at 4 °C overnight. After washes in TBS-T, membranes were incubated with the horseradish peroxidase secondary antibody at room temperature for 1 h. The immunoreactive bands were visualized by enhanced chemiluminescence with the ECL system (Amersham Biosciences) according to the manufacturer instructions. The levels of protein expression were quantified densitometrically and normalised against their respective expressions of β-actin. Prestained protein markers were used as molecular weight standards for each analysis.
2.3
Immunohistochemistry
At each gestational age three placentas were fixed by immersion in 4% paraformaldehyde overnight, dehydrated and embedded in paraffin wax. Sections (6 μm thick) were dewaxed and immunostained according to our standard protocol [21]. When necessary, antigen retrieval was performed in a pressure cooker for 2 min in 0.01 M Citrate buffer at pH 6. Primary antibodies (Table 1), diluted in goat serum, were applied overnight in a humidified chamber at 4 °C. Binding was detected using Vectastain Elite ABC kits (Vector Laboratories) and SigmaFast DAB (Sigma), according to the manufacturers’ instructions. Sections were then lightly counterstained with haematoxylin. Negative controls were performed by omitting incubation with the primary antibody. For each antibody slides from all the gestational ages were prepared and immunostained in the same batch, ensuring identical conditions for comparisons.
In order to localise expression of the antigens to specific cell types staining intensity was graded visually from 0–3, where 0 represented the negative control. Three people scored the sections independently, blinded to the gestational age or experimental protocol, and the mean value was taken.
2.4
Immunofluorescent dual-labelling
Dual immunofluorescent labelling was performed in order to test for co-localisation of markers of oxidative stress and COX expression in individual cells. Sections were dewaxed, permeabilised in TBS containing Triton X-100 (0.1%) and Tween 20 (0.1%) (TBS-TT) for 30–60 min, and blocked in 5% bovine serum albumin for 20 min at room temperature. Primary antibodies diluted in TBS-TT were applied overnight at 4 °C. Negative control sections had primary antibodies omitted. After three 10-min washes in TBS-TT, sections were incubated for 1 h at room temperature with a mixture of fluorescent secondary antibodies, containing anti-goat Alexa 488 and anti-rabbit Alexa 568 (both used 1/100; from Molecular Probes) in TBS-TT. Sections were washed in TBS-TT as before and then twice in distilled water for 5 min and subsequently mounted in Vectashield mounting medium containing DAPI (Vector, UK). Images were captured using a Leica confocal microscope (LeicaTCS-NT, Leica Instruments GmbH).
Co-localisation was identified in overlay images by a yellow signal caused by the combination of the green signal (COX-1/COX-2) and the red signal (HNE).
2.5
TUNEL assay
Staining was performed using In-Situ Cell Death Detection kit-Texas Red (Roche Applied Science) according to the manufacturer's instructions. Conditions were adjusted so that the staining pattern matched morphological assessments of apoptosis based on examination of resin-embedded samples. Once the protocol had been optimised an apoptotic index was calculated as follows:
2.6
Dual-fluorescence TUNEL assay & Active Caspase-3, and HNE co-localisation
Staining was performed using In-Situ Cell Death Detection kit-Texas Red (Roche Applied Science) according to the manufacturer's instructions, and using an Anti-Active Caspase-3 antibody (Promega) at 1:50, and Anti-HNE as previously at 1:200. Samples were also DAPI stained to intercalate the DNA and fluoresce cell nuclei. For negative controls, omission of primary antibody and TdT enzyme were performed separately. For positive controls, post-lactational mouse mammary glands were used. For confocal microscopy, all samples were analysed during one session to avoid bias, and multiple (usually 5) fields of view per labyrinth were saved for further analysis.
2.7
Ex-vivo culture experiment
In order to induce oxidative stress placental samples were subjected to hypoxia-reoxygenation (H/R) in vitro [22]. Placentas were taken from 3 animals at gestational age E18, halved on an ice block and immersed in ice-cold PBS prior to culture. Three samples, each 5–10 mm3, from each placenta were incubated in culture medium (TCS large vessel endothelial cell basal medium (TCS CellWorks, Milton Keynes, UK)), which had been maintained for several hours in the specified gas composition at 37 °C. Data for intraplacental oxygen concentrations are not yet available for the mouse, and so estimates were made on the basis of results for another small mammal, the guinea-pig. The umbilical venous pO2 towards term is 29.5 mmHg, which is close to that for fetal primates and ungulates [23]. Therefore, for normoxia a gas mix of 5% O2, 90% N2, 5% CO2 was used, whereas for hypoxia it was 1% O2, 94% N2, 5% CO2. Gas composition was monitored and controlled throughout using Biospherix (Redfield, NY) Pro-Ox and Pro-CO2 probes. Control samples were cultured for 4 h in normoxia, whereas samples subjected to H/R experienced 1 h of hypoxia followed by 3 h of normoxia. Following culture the tissues were either frozen or fixed for Western blotting and immunohistochemistry respectively.
2.8
Statistical analyses
Statistical significance was calculated by using one-way analysis of variance (ANOVA). Differences between groups were assessed using Fisher's Least Significant Difference Test. Statistical significance was assumed at a P-value <0.05.
3
Results
3.1
Markers of oxidative stress
Hydroxynonenal (HNE) is a marker of lipid peroxidation. Western blotting revealed significant changes across gestational age (P = 0.003), with low concentrations of HNE at E14, a significant increase at E16, and a subsequent decline by E18 (Fig. 1a,b).
Scoring based on IHC revealed different intensities of immunoreactivity in the different cell populations, and different temporal patterns. At E14 immunoreactivity was low in all cell types (Fig. 1c,d). At E16 there was moderate staining (scored 2) in the syncytiotrophoblast of the LZ, which peaked (3) at E18 and declined to 1 by E19 (Fig. 1e). The cytotrophoblast cells of the LZ stained uniformly at level 1 throughout, and no immunoreactivity was observed in the fetal endothelial cells. In the JZ strong staining was observed in the GCs at E16 (2.5) and E18 (3) (Fig. 1f), and this was maintained at 2.5 at E19. The spongiotrophoblast cells were less immunoreactive, scoring only 0.5 throughout gestation, and no immunoreactivity was observed in the giant cells or the decidua.
Nitrotyrosine (NT) staining indicates the formation of the prooxidant peroxynitrite, and of an imbalance in the production of the superoxide ions and nitric oxide. Immunostaining revealed a similar pattern to HNE, with almost undetectable staining in early samples and significant increases from day 16 onwards (data not shown).
Therefore, oxidative stress increases during gestation in the murine placenta, specifically in syncytiotrophoblast and glycogen cells.
3.2
COX-1
Western blotting revealed significant changes in concentrations of COX-1 across gestational age (P = 0.013). There was an increase from E14 to E16, followed by a significant decrease by E18 (Fig. 1a,b).
At E14 COX-1 immunoreactivity was not observed in any cell type, except for low intensity (level 1) staining in the GCs. At E16 strong reactivity (3) was visible in the GCs, with lower intensity staining (1) being present in the spongiotrophoblast and cytotrophoblast cells (data not shown). By E18 there was a general increase in staining, with many of the cytotrophoblast cells (2) reacting positively, along with the syncytiotrophoblast (2), and all GCs (3) (Fig. 2a,b). This pattern persisted at E19, when the decidua (2) was also immunoreactive. By contrast, the spongiotrophoblast cells displayed low (0.5) immunoreactivity throughout gestation.
Towards term COX-1 and HNE immunoreactivity were present within the same cells in both the LZ and JZ, as evidenced by the dual-labelling experiments (Fig. 2c,d).
3.3
COX-2
Western blotting revealed a significant change in concentrations of COX-2 across gestation (P = 0.002), peaking at E19 (Fig. 1).
IHC demonstrated that COX-2 expression is low (level 1) within the syncytiotrophoblast, spongiotrophoblast and GCs at E14. With advancing gestational age immunoreactivity increased, principally in the cytotrophoblast cells (2), GCs (2.5) and spongiotrophoblast (1) at E18 and E19 (Fig. 3a,b). The syncytiotrophoblast displayed low immunoreactivity (0.5) throughout gestation.
Again there was strong co-localisation between immunoreactivity for COX-2 and HNE (Fig. 3c).
3.4
Detection of apoptotic changes
During apoptosis cytokeratins within trophoblast cells are cleaved by active-caspase 3 to yield a specific product detected by the M30 antibody [24]. The labyrinth showed low immunoreactivity for active-caspase 3 until E18 (Fig. 4b), but thereafter it increased in intensity until E19. The staining was localised to both the syncytiotrophoblast and individual cytotrophoblast cells.
Immunoreactivity for M30 also increased with gestational age, and was most abundant at E18, declining slightly at E19 (data not shown).
In order to estimate the number of apoptotic nuclear profiles TUNEL staining was performed and an apoptotic index calculated. TUNEL-positive nuclei were first observed at E16 when the apoptotic index was 2.7%, and steadily increased in frequency until E19, when the index reached 20.0% (P < 0.001). There was a strong temporal and spatial association between TUNEL staining and immunoreactivity for HNE (Fig. 4a) and active caspase-3 (Fig. 4b). At E16 a number of cells were caspase-positive but TUNEL-negative. Later, almost all TUNEL-positive nuclei were surrounded by cytoplasm immunoreactive for active caspase 3. TUNEL-positive nuclei were rarely observed in the absence of caspase-positivity, when they were considered to be necrotic.
To further confirm trophoblast cells were undergoing apoptosis, morphological evidence was sought by examining resin embedded samples. Cells displaying peripheral nuclear chromatin condensation, some in the classical crescent shape, were seen with increasing frequency towards E19 (Fig. 4c). There was occasional evidence of apoptotic body formation, but other classical characteristics, such as plasma membrane blebbing, could not be resolved with light microscopy. The position of the nuclei suggested that the majority was within the syncytiotrophoblast, but the involvement of cytotrophoblast and endothelial cells could not be excluded at the light microscopic level.
3.5
Ex-vivo culture experiments
To test the relationships between oxidative stress and the induction of COX enzymes and apoptosis experimentally, placental explants were subjected to hypoxia/reoxygenation (H/R), a known stimulus for the generation of reactive oxygen species [22].
Western blotting confirmed increased concentrations of HNE and COX-1 in the explants following H/R compared to control samples frozen at the start of the experiment (time zero) or cultured under normoxic conditions. For COX-2 a significant increase was only observed compared to the time zero controls (Fig. 5a,b).
IHC localised the HNE to individual cytotrophoblast cells and to the syncytiotrophoblast layers of the labyrinth. In the JZ individual spongiotrophoblast cells were immunopositive, but the strongest staining was seen in the GCs within the JZ and decidua. Dual-labelling revealed strong co-localisation for COX-1 and COX-2 in the spongiotrophoblast and GCs in the JZ, and within the syncytiotrophoblast and cytotrophoblast cells in the labyrinth (Fig. 5c).
IHC revealed increased immunoreactivity for M30 within the trophoblast following H/R. The apoptotic index based on TUNEL labelling increased from 2.8% and 1.6% in the time zero and normoxic controls respectively, to 20.6% following H/R (P = 0.006). The majority (78%) of TUNEL positive cells were also immunoreactive for HNE on dual-labelling (Fig. 5d). From resin-embedded material it appeared that most of the cells with condensed peripheral chromatin following H/R were within the syncytiotrophoblast layers (data not shown).
From these data it can be concluded that there is a significant increase in oxidative stress following H/R, and that this is associated with increased expression of COX-1 and COX-2 expression in the LZ and JZ, and with increased apoptosis in the labyrinth.
4
Discussion
These results indicate that oxidative stress increases with gestational age in the murine placenta during normal pregnancies, and that it may play a significant physiological role by inducing higher concentrations of the COX-1 and COX-2 enzymes, and hence increasing prostaglandin synthesis. Our data also indicate a close temporal and spatial association between oxidative stress and trophoblast apoptosis within the labyrinth. Trophoblast apoptosis has been linked to the pathophysiology of preeclampsia, and the mouse might provide a useful genetic model in which to elucidate the mechanisms underlying the shedding of apoptotic debris.
We have recently proposed that placental oxidative stress arises through fluctuations in oxygenation [3,25], and its development in the murine placenta towards term is consistent with this view. As fetal and placental oxygen extraction reach their peaks so any transient mismatch between maternal oxygen supply and feto-placental demand will lead to a dip in the intraplacental oxygen concentration. The fact that H/R in vitro is a powerful inducer of oxidative stress, and that the stress localises to the same tissues as in late gestation supports this hypothesis. It is now accepted that oxidative stress induces a spectrum of cellular changes, ranging from the physiologically homeostatic to the frankly pathologic. Induction of enzymes is towards the more physiological end of that spectrum, whereas apoptosis is towards the opposite extreme.
Induction of the COX enzymes has been linked to oxidative stress through activation of the p38MAPK and the NF-κB family of transcription factors in other cell types [26–30]. In our study there appeared to be strong co-localisation between the formation of HNE and expression of COX-1 and -2 to individual cytotrophoblast, spongiotrophoblast and glycogen cells, both in vivo and in vitro. However, there was an apparent discrepancy between the Western blot results and our IHC findings, for the former indicated a peak in oxidative stress and COX-1 at E16 while the staining intensity for HNE and COX-1 increased until E19. This difference can be accounted for by the fact that the number of GCs declines by about half between E16 and E18 [31], so reducing their contribution to the overall tissue homogenate.
Expression of the COX enzymes appears to be mainly altered in placental rather than decidual tissues. Changes with gestational age were observed in the trophoblast populations within the LZ, but the greatest changes in immunoreactivity were seen within the GCs and the spongiotrophoblast cells in the JZ. Expression of COX-1 and COX-2 has recently been reported in the rat placenta, and that of COX-2 similarly increases with gestational age, particularly in the JZ [32]. The placental cells in this zone have direct access to maternal blood as they line the maternal venous sinuses. Therefore, it is possible that PGs synthesized in this zone in response to oxidative stress are released into the maternal circulation. It is also notable that the GCs migrating into the DB are strongly immunoreactive for COX, and so PGs synthesised by these cells could exert local paracrine effects in the decidua. As PGs have been linked with the induction of parturition [10,11,33] this raises the possibility that placental oxidative stress could play a role in initiating or augmenting uterine contractions should the mismatch in maternal blood supply and feto-placental demands become extreme.
Trophoblast apoptosis increased late in gestation as assessed by IHC for M30 and TUNEL, and cell morphology. There was a close temporal and spatial co-localisation with oxidative stress, although on Western blotting evidence of oxidative stress appeared to decline in the last days of gestation. This may again reflect changes in the cell populations within the placenta and associated decidua. The apoptotic index based on TUNEL-positivity showed a continual increase until term, although M30 immunoreactivity suggested a decline after E19. There is strong evidence that following initial cleavage, exposing the M30 epitope, cytokeratin 18 becomes further cleaved to smaller fragments [24]. In these late stages of apoptosis the M30 epitope may be lost, and so the antibody no longer detects all apoptotic trophoblast [34]. A causal link between oxidative stress and apoptosis was strengthened by the fact that apoptosis could be induced by H/R in vitro. As in the human placenta this was associated with increased concentrations of active caspase-3 [20]. The mitochondrial pathway was strongly implicated in the apoptotic process in the human, but further work is required to determine whether the same mechanism operates in the mouse.
The increase in oxidative stress and apoptosis in the LZ with gestational age may explain some of the growth dynamics of the murine placenta. Overall placental volume increases with gestational age until E16.5 and then plateaus, coinciding with the rise in oxidative stress. It is notable that within the LZ the volume and surface area of the trophoblast plateaus at E16.5 whereas those of the fetal capillaries continue to increase until E18.5 [35]. The fetal endothelial cells did not display immunoreactivity for HNE or nitrotyrosine, and did not appear to label with TUNEL, suggesting that oxidative stress and its consequences are lower in this cell type.
The links between oxidative stress and apoptosis suggest that the mouse may provide a useful genetic model in which to investigate the relative roles of antioxidant enzymes and signalling pathways in regulating trophoblast apoptosis. Apoptosis increases towards term in normal human pregnancies [36], but increased rates have been reported in preeclamptic placentas and in placentas from cases of intrauterine growth restriction [37–39]. It is thought that microparticles of apoptotic, or aponecrotic, debris released from the apical surface of the syncytiotrophoblast into the maternal circulation result in an enhanced maternal inflammatory response in preeclampsia [6]. Whether apoptotic debris is shed from the murine labyrinth as in the human placenta towards the end of gestation remains to be determined.

oxidative stress
cyclooxygenases
apoptosis
murine placenta