S Hackett

P Pootrakul

Professor Tim G St

Pierre
email: stpierre@physics.uwa.edu.au

The magnetic susceptibilities of iron deposits in thalassaemic spleen tissue

Keywords: haemosiderin, ferritin, magnetic susceptibility, thalassaemia, iron

The iron-specific magnetic susceptibility of tissue iron deposits is used in the field of non-invasive measurement of tissue iron concentrations. It has generally been assumed to be a constant for all tissue and disease types. The iron-specific magnetic susceptibilities χ Fe for spleen tissue samples from 7 transfusion dependent βthalassaemia ( β-thal) patients and 11 non-transfusion dependent βthalassaemia/Haemoglobin E (β/E) patients were measured at 37°C. Both groups of patients were iron loaded with no significant difference in the distribution of spleen iron concentrations between the two groups. There was a significant difference between the mean χ Fe of the spleen tissue from each group. The non-transfusion dependent β/E patients had a higher mean (± standard deviation) spleen χ Fe (1.55 ± 0.23 × 10 -6 m 3 /kg Fe) than the transfusion dependent β-thal patients (1.16 ± 0.25 × 10 - 6 m 3 /kg Fe). Correlations were observed between χ Fe of the spleen tissue and the fraction of magnetic hyperfine split sextet in the 57 Fe Mössbauer spectra of the tissues at 78 K (Spearman rank order correlation r = -0.54, p = 0.03) and between χ Fe of the spleen tissue and the fraction of doublet in the spectra at 5 K (r = 0.58, p = 0.02) indicating that χ Fe of the spleen tissue is related to the chemical speciation of the iron deposits in the tissue.

Introduction

Iron overload is a condition that affects patients with haemoglobinopathies who either require regular blood transfusions [START_REF] Brittenham | Clinical consequences of new insights in the pathophysiology of disorders of iron and heme metabolism[END_REF] or have increased dietary iron absorption, and also patients with various forms of hereditary hemochromatosis [START_REF] Pietrangelo | Hereditary hemochromatosis -a new look at an old disease[END_REF]. Measurement of tissue iron concentrations is a part of the clinical management of many of these patients [START_REF] Brittenham | Noninvasive methods for quantitative assessment of transfusional iron overload in sickle cell disease[END_REF]. Liver iron concentration (LIC) is considered to be a reliable indicator of overall body iron burden. However, direct measurement of LIC by chemical analysis of needle biopsy samples is problematic both because of the sampling error introduced by the small size of the sample obtained and because of the associated morbidity and mortality risks associated with biopsy [START_REF] Villeneuve | Variability in hepatic iron concentration measurement from needle-biopsy specimens[END_REF][START_REF] Kreeftenberg | Measurement of iron in liver biopsies -a comparison of three analytical methods[END_REF][START_REF] Ambu | Uneven hepatic iron and phosphorus distribution in β-thalassemia[END_REF][START_REF] Emond | Quantitative study of the variability of hepatic iron concentrations[END_REF]. Over the past two decades or so, non-invasive methods of measurement of LIC have been developed which exploit the large and positive magnetic susceptibility of tissue iron deposits relative to the small and negative magnetic susceptibility of the other components of body tissues [START_REF] Farrell | An application of superconductivity to medical diagnostics[END_REF][START_REF] Fischer | Liver iron susceptometry[END_REF][START_REF] Brittenham | Non-invasive in vivo measurement of human iron overload by magnetic susceptibility studies[END_REF][START_REF] St | Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance[END_REF]. The iron deposits in iron loaded tissue are predominantly in the form of haemosiderin and ferritin [START_REF] O'connell | The role of iron in ferritin-and haemosiderin-mediated lipid peroxidation in liposomes[END_REF]. Haemosiderin is an insoluble pathological compound containing particles of hydrated iron(III) oxyhydroxide associated with organic components while ferritin is an iron storage and homeostasis protein [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF].

Ferritin is composed of 24 polypeptide subunits that self assemble to form an approximately spherical shell-like structure with an external diameter of approximately 12 nm and an internal cavity diameter of approximately 8 nm [START_REF] Harrison | The structures of ferritin and apoferritin: some preliminary xray data[END_REF].

Iron is stored within the cavity in the form of a particle of hydrated iron(III) oxyhydroxide. Although both ferritin and haemosiderin have an inorganic iron(III) oxyhydroxide component, the chemical speciation of this component can vary. Three different chemical species of tissue iron(III) oxyhydroxide have been identified oxyhydroxide [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Mann | Structural specificity of haemosiderin iron cores in ironoverload diseases[END_REF][START_REF] Dickson | Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins[END_REF]. The chemical speciation of these iron(III) oxyhydroxides has been shown to be different in different identifiable groups of iron loaded patients [START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai β-thalassemia/hemoglobin E patients and Australian β-thalassemia patients[END_REF]. Since different chemical speciation of iron oxides is known to lead to different iron-specific magnetic susceptibilities [START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF], a knowledge of the magnetic susceptibilities of ferritins and the different forms of haemosiderin is of practical importance in the field of non-invasive measurement of tissue iron concentrations using methods such as biomagnetic susceptometry and magnetic resonance imaging that exploit the relatively large magnetic susceptibility of these materials.

The magnetic susceptibilities of ferritin and haemosiderin were first measured by Michaelis and coworkers [START_REF] Michaelis | The magnetic properties of ferritin and some other colloidal ferric compounds[END_REF]. They made measurements on samples of horse spleen ferritin at 27°C and haemosiderin at 2 2°C yielding iron specific magnetic susceptibility values of 1.33 (± 0.02) x 10 -6 m 3 /kg Fe for the ferritin and 1.1 x 10 -6 m 3 /kg Fe for the haemosiderin. This work was later followed by measurements at 23°C on human ferritin and haemosiderin isolated from liver tissue by Schoden and Sturgeon [START_REF] Shoden | Hemosiderin I. A Physico-Chemical Study[END_REF] yielding values of 1.6 x 10 -6 m 3 /kg Fe for the ferritin and values of 1.5, 2.1, and 1.2 × 10 -6 m 3 /kg Fe for three different human haemosiderin samples. The value of 1.6 × 10 -6 m 3 /kg Fe for human ferritin measured by Schoden and Sturgeon has subsequently been used by Fischer and coworkers [START_REF] Fischer | Assessment of iron stores in children with transfusion siderosis by biomagnetic liver susceptometry[END_REF] to obtain non-invasive in vivo measurements of liver iron concentrations in iron loaded patients using the technique of magnetic biosusceptometry [START_REF] Bauman | Estimation of hepatic iron stores by vivo measurement of magnetic susceptibility[END_REF][START_REF] Messer | Magnetic susceptibility and hepatic iron stores: the correlation betweenquantitative determinations of susceptibility and iron in human liver specimens[END_REF]. This value is now routinely used in biosusceptometry measurements to determine liver iron concentrations. However, the

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biological variability in the magnetic susceptibility measurements of Michaelis and coworkers [START_REF] Michaelis | The magnetic properties of ferritin and some other colloidal ferric compounds[END_REF] and Schoden and Sturgeon [START_REF] Shoden | Hemosiderin I. A Physico-Chemical Study[END_REF] appears to be larger than their reported analytical variability suggesting that the magnetic susceptibility of tissue iron deposits may vary between individuals. Furthermore, since the early measurements of the magnetic susceptibilities of ferritin and haemosiderin, there has been the discovery that there are different chemical species of iron(III) oxyhydroxide associated with different samples of haemosiderin and that the different chemical species have different magnetic properties at cryogenic temperatures [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Dickson | Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins[END_REF][START_REF] Allen | Lowfrequency low-field magnetic susceptibility of ferritin and hemosiderin[END_REF].

These observations raise the questions of whether or not there are significant differences in the body temperature magnetic susceptibilities of the different chemical species of tissue iron deposit and to what degree these differences might affect measurements of tissue iron concentration by biomagnetic susceptometry techniques when different species are present or when the chemical speciation is unknown.

In order to address these questions, the iron specific magnetic susceptibilities of iron loaded spleen tissues from two identifiably different groups of iron-loaded patients have been measured. Spleen tissue was chosen because it is readily available after splenectomy procedures. The two groups of patients have been shown in a previous study to have different chemical speciation of the spleen tissue iron deposits [START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai β-thalassemia/hemoglobin E patients and Australian β-thalassemia patients[END_REF].

The characterisation of the chemical speciation of the iron in these tissues is extended in this study. A sample of human liver ferritin was also prepared to enable comparison of results with those on human liver ferritin measured by Schoden and Sturgeon [START_REF] Shoden | Hemosiderin I. A Physico-Chemical Study[END_REF]. The ferritin was isolated from four livers obtained post-mortem from β-thalassaemia/Hb E subjects according to the method of ferritin purification described in Biomineralisation [START_REF] Chua-Anusorn | Pathological biomineralization of iron[END_REF]. These subjects had received few, if any, blood transfusions.

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Materials and Methods

Mössbauer spectra were recorded in transmission geometry using a 57 Co in rhodium foil source. The source was driven at constant acceleration from -13 to +13 mm/s with a double ramp waveform. Spectra were subsequently folded to eliminate the parabolic background caused by variation in the solid angle subtended by the detector window about the source. The resulting spectra consisted of 250 data points with a background count of between approximately 1 × 10 6 and 2 × 10 6 per channel. The counting time was determined by the iron concentration of the sample under study.

The velocity scale was calibrated with reference to the spectrum of an α-iron foil at room temperature, the centre of the spectrum being taken as zero velocity. Freeze dried samples of tissue were packed into 10 mm diameter Perspex sample holders for the 78 K spectra and 13 mm copper sample holders with Mylar windows for the 5 K measurements. The thickness of the sample was adjusted so that the 14.4 keV Mössbauer γ rays were attenuated by approximately 1/e. Sample temperatures were maintained at 78 K or at 5 K during the spectral data acquisition using a liquid nitrogen or liquid helium cryostat respectively.

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Each Mössbauer spectrum was initially fitted with a doublet and sextet of Lorentzian peaks using a sum of squares minimization procedure. In some spectra, a small signal owing to haem-iron was detectable and as such a second doublet was included in the fitting procedure. The doublet from haem-iron is clearly distinguishable from that of paramagnetic haemosiderin iron since it exhibits a much larger quadrupole splitting giving a clearly resolved peak even when superimposed on a paramagnetic haemosiderin doublet [START_REF] Dickson | Applications to biological systems[END_REF]. In such cases, the relative spectral area of the haem doublet was used to measure the fraction of haem-iron in the sample. The two peaks in each doublet were constrained to be of equal area and width while the ratio of areas of the outer to middle to inner pairs of peaks of the sextet were constrained to be in the ratio 3:2:1. The linewidths, centre shift, and quadrupole splitting of the doublet were allowed to vary freely during the fits as were the linewidths, magnetichyperfine-field splitting, centre shift, and quadrupole perturbation on the sextet. This fitting procedure yields reproducible spectral parameters and doublet to sextet area ratios for spectra with high signal to noise ratios. However, for some of the 78-K spectra with low sextet-signal to noise ratios, this fitting procedure tends to give unreliable results.

Mean values for the Mössbauer spectral parameters of the 78-K sextet signal in the 4 spleen spectra with the highest sextet-signal to noise ratio were used as a standard with which to refit all of the spleen tissue 78-K spectra. All of the spleen 78-K spectra were refitted with a high-spin Fe(III) doublet, a haem doublet where necessary, and the standard sextet. During the fitting procedure, the linewidths, magnetic-hyperfine-field splitting, centre shift, and quadrupole perturbation of the sextet were allowed to vary by up to one standard deviation from the mean values

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derived from the 4 spectra with the highest sextet-signal to noise ratios. The area of the sextet was allowed to vary freely. In this way the relative spectral area of the sextet in spectra with very low sextet-signal to noise ratios could be more reliably assessed assuming that the Mössbauer spectral parameters of the sextet do not vary significantly from sample to sample.

Samples of the freeze-dried powders (both tissues and ferritin sample) were compressed into pellets of approximate mass 60 mg for magnetic susceptometry measurements. All pellets were less than 4.5 mm in length. For three of the spleens, duplicate pellets were made in order to test reproducibility of measurements. Pellets were mounted in long uniform plastic tubes and were held in place in the tubes for magnetometry measurements with plastic collars of less than 8mm in length that surrounded the pellets symmetrically. The tubes were much longer than the dimensions of the sense coils and hence do not contribute to the measured signal.

However, the diamagnetic susceptibility of the collars was measured independently and accounted for in the measurement of the sample magnetic susceptibilities by subtraction of the moment of the collar from the moment of the collar and pellet.

Magnetic susceptometry measurements were made using a Quantum Design magnetic properties measurement system (MPMS-7). The instrument uses a sensor based on a superconducting quantum interference device (SQUID) for detection of sample magnetic moments as they are passed through a second-order gradiometer superconducting sensor coil system. The sensor coil system consists of a central coil Sample magnetic moments were measured as a function of applied magnetic field over the range of fields ± 2.4 x 10 6 A/m (± 30 kOe). Sample temperatures were maintained at 37°C during the measurements. At this temperature and over this magnetic field range the magnetic moment of the samples was approximately linear with applied magnetic field, consistent with previous observations of magnetic susceptibility of ferritin at temperatures close to body temperature [START_REF] Makhlouf | Magnetic hysteresis anomalies in ferritin[END_REF][START_REF] Brooks | Relaxometry and magnetometry of ferritin[END_REF]. Ironspecific magnetic susceptibilities were determined by measuring the slope of the mass-specific magnetic moment against applied magnetic field and correcting for collar magnetic susceptibility and iron-free-tissue magnetic susceptibility (see Results

section).

Tissue samples were stored in the freeze dried state between measurement of Mössbauer spectra and measurement of the magnetic susceptibility. In order to check that no change in chemical speciation had occurred during the intervening period, a

Mössbauer spectrum was recorded for a second time at 78K for a sample of spleen tissue with a mixture of spectral species after the magnetic susceptibility measurement. No changes in the Mössbauer spectral parameters were detectable indicating that there had been no detectable change in the chemical speciation of iron within the sample during the period of the study.

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Results

Elemental analysis of the tissue samples indicated tissue iron concentrations ranging from 2.2 to 21 mg Fe g -1 dry tissue. The means (± standard deviation) of the nonhaem iron concentration of the β-thalassaemic and β-thalassaemia/Hb E samples were 7.7 (± 6.3) and 5.6 (± 1.9) mg/g dry tissue, and the medians (and interquartile ranges) were 6.1 (3.6 -8.9) and 5.7 (4.4 -9.3) mg/g dry tissue respectively. There was no significant difference in the distribution of tissue iron concentrations between the two groups of patients as determined by a Wilcoxon-Mann-Whitney (W-M-W) test (p = 0.93).

Mössbauer Spectroscopy

Mössbauer spectra of the spleen tissue samples comprised a superposition of a doublet spectral component (attributed to paramagnetic or superparamagnetic highspin iron(III) ) and a sextet spectral component (attributed to magnetically blocked iron(III) ) at both 78 K and 5 K (see Figure 1). For an explanation of terms and physical principles related to the interpretation of the Mössbauer spectra of tissue iron deposits, see St Pierre et al [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF]. For some samples a small doublet attributable to haem iron was also apparent (1 spleen from a transfusion dependent β-thalassaemia patient showed a 20% haem doublet; 2 spleens from non-transfusion dependent βthalassaemia/Hb E patients showed 6% and 1% haem doublets each; for all other samples a haem doublet was not detectable). Mean values for the fitted parameters of each spectral component are given in Table 1. The fraction F S of the non-haem

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spectral signal in the form of a sextet at 78 K is related to the fraction of non-haem tissue iron in the form of the goethite-like iron oxyhydroxide [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Mann | Structural specificity of haemosiderin iron cores in ironoverload diseases[END_REF][START_REF] Dickson | Mössbauer spectroscopic studies of haemosiderins from different sources[END_REF]. The mean (± standard deviation) of F S for spleens from the transfusion dependent βthalassaemia patients was 0.27 ± 0.12 and from the non-transfusion dependent βthalassaemia/Hb E patients was 0.129 ± 0.036. The medians (and interquartile ranges) of F S were 0.26 (13 -33) and 0.125 (0.102 -14.1) for the β-thalassaemia and β-thalassaemia/Hb E patients respectively. The distributions of F S for the two patient groups were significantly different (W-M-W test p = 0.01) (Figure 2). The fraction F D of non-haem spectral signal in the form of a doublet at 5 K is reported to be related to the fraction of tissue non-haem-iron in the form of non-crystalline iron oxyhydroxide [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Mann | Structural specificity of haemosiderin iron cores in ironoverload diseases[END_REF][START_REF] Dickson | Mössbauer spectroscopic studies of haemosiderins from different sources[END_REF]. The mean (± standard deviation) of F D for spleens from the transfusion dependent β-thalassaemia patients was 0.22 ± 0.08 and from the nontransfusion dependent β-thalassaemia/Hb E patients was 0.43 ± 0.19. The medians (and interquartile ranges) of F D were 0.26 (0.18 -0.36) and 0.41 (0.25 -0.59) for the β-thalassaemia and β-thalassaemia/Hb E patients respectively. The distributions of F D for the two patient groups were not found to be significantly different using a W-M-W test. A statistically significant correlation was found between F S and tissue non-haem-iron concentration but not between F D and tissue non-haem-iron concentration. A weak correlation was found between F S and F D (Spearman rank order correlation coefficient 0.47, p = 0.051). The relationship between the two parameters appears non-linear (Figure 3) with F S remaining at approximately 0.10 until F D drops below approximately 0.35. As F D drops below 0.35 there is a very rapid rise in F S to values around 0.40.

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Magnetic Susceptibility Measurements

The mass-specific magnetic susceptibility, χ Mass , of each tissue sample is shown plotted against tissue non-haem-iron concentration in Figure 4. The relationship between mass-specific magnetic susceptibility and tissue non-haem-iron concentration appears to be different for the two patient groups with the transfusiondependent β-thalassaemia patients exhibiting a generally lower non-haem-ironspecific magnetic susceptibility, χ Fe , than the non-transfusion-dependent βthalassaemia/Hb E patients. The mass-specific magnetic susceptibility and nonhaem-iron concentration data were fitted with the following model, based on Weidemann's mixture equation

χ Mass = ′ χ Fe × Fe [ ] + χ Tissue × 1 -Fe [ ] ( ) , (1)
where χ Tissue is the mass-specific magnetic susceptibility of non-haem-iron-free spleen tissue and [Fe] is the tissue non-haem-iron concentration expressed as the fraction of the mass of the tissue that is non-haem-iron. Fits of this model to the data from each patient group are shown in Figure 4 yielding average non-haem-iron-specific magnetic susceptibilities (χ′ Fe ) of 1.10 ± 0.10 × 10 -6 m 3 /kg Fe and 1.60 ± 0.20 × 10 -6 m 3 /kg Fe and mass-specific magnetic susceptibilities for non-haem-iron-free tissue (χ Tissue ) of -7.8 ± 1.0 × 10 -9 m 3 /kg dry tissue and -7.9 ± 1.3 × 10 -9 m 3 /kg dry tissue for the transfusion-dependent β-thalassaemia and non-transfusion-dependent βthalassaemia/Hb E patients respectively. It should be noted that the values of the average non-haem-iron-specific magnetic susceptibilities deduced from this model are based on the assumption that there is no increase in concentration of other high magnetic susceptibility elements concomitant with the non-haem-iron concentration

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increases. The validity of this assumption is supported by a previous study of macro, trace, and ultra-trace elements in thalassaemic tissues [START_REF] Shuler | Effect of thalassemia/hemoglobin E disease on macro, trace, and ultratrace element concentrations in human tissue[END_REF].

The values for the mass-specific magnetic susceptibilities of non-haem-iron-free tissue (χ Tissue ) from the above analysis were used to calculate a value of the nonhaem-iron-specific magnetic susceptibility (χ Fe ) for each individual tissue specimen using the following equation

χ Fe = χ Tissue + χ Mass -χ Tissue Fe [ ] . ( 2
)
The distributions of values of χ Fe for each patient group are shown in Figure 6 shows the values of χ Fe for each tissue specimen plotted against F S . There is a statistically significant correlation between χ Fe and F S (Spearman rank order correlation coefficient -0.54, p = 0.03) with higher values of F S yielding generally

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lower values of χ Fe . Figure 7 shows the values of χ Fe for each tissue specimen plotted against F D . There is a statistically significant correlation between χ Fe and F D (Spearman rank order correlation coefficient 0.58, p = 0.02) with higher values of F D yielding generally higher values of χ Fe .

The iron-specific magnetic susceptibility of the pooled human liver ferritin sample was measured by assuming that the mass-specific magnetic susceptibility of the noniron component of ferritin is equivalent to the average mass-specific magnetic susceptibilities of non-haem-iron-free spleen tissues (χ Tissue ) -7.8 × 10 -9 m 3 kg. It is worth noting that even a reasonably large percentage error on the value of the massspecific magnetic susceptibility of the non-iron component of ferritin has very little effect on the result obtained for the iron-specific magnetic susceptibility of ferritin.

The value of χ Fe for the ferritin sample was found to be 1.14 (±0.11) × 10 -6 m 3 kg Fe -1 which is consistent with the value of 1.33 (± 0.02) × 10 -6 m 3 kg Fe -1 obtained for horse spleen ferritin [START_REF] Michaelis | The magnetic properties of ferritin and some other colloidal ferric compounds[END_REF] but is significantly less than the value of 1.64 (±0.04) × 10 -6 m 3 kg Fe -1 obtained by Shoden & Sturgeon [START_REF] Shoden | Hemosiderin I. A Physico-Chemical Study[END_REF] for human liver ferritin.

Measurement of χ Fe for three duplicate samples of homogenised spleen tissue indicated an overall analytic variability of approximately 9.6%.

A C C E P T E D M A N U S C R I P T Discussion

The spleen samples from the two groups of patients covered a range of tissue nonhaem-iron concentrations but the distributions of non-haem-iron concentration in the two groups were similar thus enabling comparisons to be made between the two groups. In all cases the predominant form of iron in the tissues was non-haem iron.

The Mössbauer spectra recorded for all of the tissues in this study consisted of a central doublet characteristic of high-spin Fe(III) superposed on a spectral sextet, the sextet being due to magnetic-hyperfine-field splitting of the 57 Fe nuclear energy levels. Similar central doublet and sextet signals have been observed in previous reports of Mössbauer spectra of human tissues or extracts from human tissues [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Dickson | Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins[END_REF][START_REF] Dickson | Mössbauer spectroscopic studies of haemosiderins from different sources[END_REF][START_REF] Webb | Biologically significant iron(III) oxyhydroxy polymers: Mössbauer spectroscopic study of ferritin and hemosiderin in pancreas tissue of βthalassemia/hemoglobin E disease[END_REF][START_REF] Webb | Biogenic Ferrihydrite: Effect of β-thalassemia/hemoglobin E disease on the structure of ferrihydrite present in ferritins isolated from iron-loaded human heart and spleen tissue[END_REF][START_REF] Ward | Further characterisation of forms of haemosiderin in iron-overloaded tissues[END_REF][START_REF] St | Core structures of haemosiderins deposited in various organs in βthalassemia/haemoglobin E disease[END_REF][START_REF] St | Mossbauer spectroscopic studies of deproteinised, sub-fractionated and reconstituted ferritins: the relationship between ferritin and haemosiderin[END_REF][START_REF] St | A Mössbauer spectroscopic study of the form of iron in iron-overload[END_REF][START_REF] Rimbert | Magnetic and quadrapolar studies of the iron storage overload in livers[END_REF][START_REF] Rimbert | Mössbauer spectroscopy study of iron overloaded livers[END_REF][START_REF] Dickson | Spin fluctuation phenomena in small particle magnetic systems: A Mössbauer spectroscopic study of collective excitations and superparamagnetic relaxation in haemosiderin[END_REF][START_REF] Bell | Mössbauer spectroscopic studies of human haemosiderin and ferritin[END_REF][START_REF] Chorazy | Mossbauer effect study of liver tissues[END_REF][START_REF] Kaufman | Nature of iron deposits on the cardiac walls in β-thalassemia by Mössbauer spectroscopy[END_REF][START_REF] Chua-Anusorn | Mössbauer spectroscopic study of the forms of iron in normal human liver and spleen tissue[END_REF]. On the basis of these previous reports these spectral components can be identified as being due to polynuclear iron(III) oxyhydroxide deposits in the tissue.

The assignment of these signals to polynuclear iron(III) oxyhydroxide deposits is consistent with the fact that the predominant form of iron found in iron loaded tissues is usually haemosiderin [START_REF] Weir | Hemosiderin and tissue damage[END_REF]. The doublet component observed in the spectrum at 78 K has parameters consistent with (a) ferritin, (b) non-crystalline haemosiderin, (c) haemosiderin based on the structure of the mineral ferrihydrite, or (d) the doublet component associated with haemosiderin based on the structure of the mineral goethite (the doublet component being due to those haemosiderin particles with a smaller magnetic anisotropy energy) [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Mann | Structural specificity of haemosiderin iron cores in ironoverload diseases[END_REF][START_REF] Dickson | Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins[END_REF]. As such it is not possible to unambiguously identify the source of this signal and the signal may be due to a combination of the above forms of iron. However, the sextet component in the spectrum at 78 K can be identified as being due to the presence of haemosiderin based on the structure of the mineral goethite since this is the only form of tissue iron deposit known to give a Mössbauer spectral sextet component with these parameters

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at 78K [START_REF] Ward | Further characterisation of forms of haemosiderin in iron-overloaded tissues[END_REF][START_REF] St | Core structures of haemosiderins deposited in various organs in βthalassemia/haemoglobin E disease[END_REF][START_REF] St | Mineralization of iron in ferritin and haemosiderin: the effect of tissue environment on mineral structure[END_REF]. Similarly, while the sextet component at 5K can be associated with any or all of ferritin, ferrihydrite based haemosiderin, and goethite-like haemosiderin, the doublet component observed at 5 K can be uniquely assigned to non-crystalline iron oxyhydroxide [START_REF] St | Ferritin and hemosiderin: structural and magnetic studies of the iron core[END_REF][START_REF] Mann | Structural specificity of haemosiderin iron cores in ironoverload diseases[END_REF][START_REF] Dickson | Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins[END_REF]. However, it is possible to rank the different forms of haemosiderin, goethite-like, ferrihydrite-like and non-crystalline iron oxyhydroxide, in order of decreasing crystallinity.

Hence, as discussed elsewhere [START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai β-thalassemia/hemoglobin E patients and Australian β-thalassemia patients[END_REF], the significantly different distributions of F S for the two groups of patients indicate that the chemical speciation of iron is different between the two groups with the transfusion dependent β-thalassaemia patients having a greater fraction of their spleen iron in the form of the goethite-like iron oxyhydroxide. In this study it has been shown that the distributions of F D for the two groups of patients are not significantly different indicating that the fraction of spleen iron in the form of non-crystalline iron oxyhydroxide is not significantly different for the two groups of patients.

The observed negative correlation and apparently non-linear relationship between F S and F D suggests that pathological conditions that favour the formation of goethite-like haemosiderin in spleen tissue hinder the formation of non-crystalline iron oxyhydroxide deposits and vice versa.

The observed linear relationships between the mass-specific magnetic susceptibility and tissue non-haem-iron concentration (Figure 4) confirm that magnetic susceptometry techniques can be used to assess tissue non-haem-iron concentrations.

However, the fact that the chemical speciation of the non-haem-iron appears to

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determine the non-haem-iron-specific magnetic susceptibility of the tissue (Figures 6 and7) suggests that errors in estimations of tissue non-haem-iron concentrations will in some part be due to the biological variability of the chemical speciation between different individuals. A likely explanation for the negative correlation between χ Fe and F s (Figure 6) is that the goethite-like deposits associated with the sextet signal in the Mössbauer spectra at 78K are more crystalline (as determined by electron diffraction measurements) and have larger particle sizes than the other forms of iron(III) oxyhydroxide tissue deposits [START_REF] Chua-Anusorn | Pathological biomineralization of iron[END_REF]. As such, there is likely to be a greater degree of spin compensation in the antiferromagnetic structures thus reducing χ Fe .

Similar arguments can be made to explain the positive correlation between χ Fe and F D (Figure 7). The doublet component in the Mössbauer spectra at 5 K is associated with non-crystalline iron(III) oxyhydroxide tissue deposits [START_REF] Chua-Anusorn | Pathological biomineralization of iron[END_REF] and hence these deposits are expected to have a greater fraction of uncompensated spins in their structures thus increasing χ Fe .

The data presented in this report are for spleen tissue. However, the organ of primary interest in the clinical use of biomagnetic susceptometry is the liver. It is currently unknown whether there is a similar magnitude of variation in the chemical speciation of tissue iron deposits in iron loaded liver tissue. Four liver samples reported in a previous study [START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai β-thalassemia/hemoglobin E patients and Australian β-thalassemia patients[END_REF] (from the group of non-transfusion dependent β-thalassaemia/Hb E patients) gave values of F S in the approximate narrow range 0.08 to 0.14. No data are available for transfusion dependent β-thalassaemia liver tissue. Samples of pancreas tissue from three non-transfusion dependent β-thalassaemia/Hb E patients did show a somewhat larger variation in F S between individuals (range from 0.12 to 0.27) [START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai β-thalassemia/hemoglobin E patients and Australian β-thalassemia patients[END_REF]. The coefficient of variation of χ Fe for all spleen specimens studied here was 21% (somewhat larger than the average analytical uncertainty on the measurement of χ Fe for an individual specimen of approximately 10%) suggesting that biological variability in χ Fe would contribute a random uncertainty of approximately 19% to non-invasive measurements of spleen non-haem-iron concentrations using biomagnetic susceptometry. If the currently accepted value of χ Fe for use in liver biosusceptometry (1.6 × 10 -6 m 3 /kg Fe ) was used to measure spleen iron concentration in the entire group of patients in this study, there would be a systematic underestimation of the tissue non-haem-iron concentration of 12.5% (SE 6%).

Further measurements of χ Fe for liver tissue specimens will need to be made in order to determine whether similar magnitudes of uncertainty would apply to biomagnetic susceptometry measurements of liver iron concentration. Differences in the ironspecific magnetic susceptibilities χ Fe for liver tissue between different patients would explain some of the variability seen in comparisons of measurements of liver iron concentration measured by non-invasive magnetic measurements with those measred by chemical assay of biopsy specimens.

  ferrihydrite (a poorly crystalline mineral with the approximate formula 5Fe 2 O 3 .9H 2 O, (ii) a mineral with a structure related to very poorly crystalline goethite (goethite has the formula α-FeOOH), and (iii) a non crystalline iron(III)

  with 2 counter-clockwise turns and 2 single clockwise turn coils 2cm above and 2cm below the central coil. As both collars and sample pellets were less than 10 mm in A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10 length they were small with respect to the coil spacing and hence were approximated as point dipoles.

Figure 5 .

 5 The mean (± standard deviation) of χ Fe for spleens from the transfusion-dependent βthalassaemia patients was 1.16 ± 0.25 ×10 -6 m 3 /kg Fe and from the non-transfusion dependent β-thalassaemia/Hb E patients was 1.55 ± 0.23 ×10 -6 m 3 /kg Fe . . The medians (and interquartile ranges) of χ Fe were 1.16 (0.85 -1.44) ×10 -6 m 3 /kg Fe and 1.63 (1.36 -1.75) ×10 -6 m 3 /kg Fe for the β-thalassaemia and β-thalassaemia/Hb E patients respectively. The distributions of χ Fe for the two patient groups were significantly different (W-M-W test p = 0.01). A statistically significant correlation was observed between χ Fe and tissue non-haem-iron concentration neither for each patient group alone nor for the two groups combined.

FIGURE 1

 1 FIGURE 1 Mössbauer spectra from a sample of β-thalassaemia/Hb E spleen tissue sample at (a) 78K and (b) 5K.

FIGURE 2 FIGURE 3

 23 FIGURE 2 Scatter plot of F S , the fraction of Mössbauer spectrum recorded at 78 K in the form of a sextet for β-thalassaemia (l) and β-thalassaemia/Hb E (s) spleen tissue

FIGURE 4 Mass

 4 FIGURE 4 Mass Susceptibility of β-thalassaemia (l) and β-thalassaemia/Hb E (s)

FIGURE 5 FIGURE 6

 56 FIGURE 5 Distribution of iron-specific magnetic susceptibilities of β-thalassaemia (l) and β-thalassaemia/Hb E (s) spleen tissue samples (p-value of 0.01).

FIGURE 7

 7 FIGURE 7 Iron-specific magnetic susceptibility of β-thalassaemia (l) and β-

This work was partially funded by Novartis Pharma. Tim St Pierre has a financial interest in and consultancy with a company, Resonance Health.

A C C E P T E D M A N U S C R I P T


C Winchester

O C Jakobs

K M Snead

Gibson

G Barcelo-Coblijn

E J Murphy

K Mills

B Winchester

C Jakobs

O C Snead

K M Gibson

Lipid abnormalities in succinate semialdehyde dehydrogenase (aldh5a1 -/-) deficient mouse brain provide additional evidence for myelin alterations

Keywords: Succinate semialdehyde dehydrogenase (SSADH), Aldehyde dehydrogenase 5a1 (Aldh5a1), γ-hydroxybutyric acid, γ-aminobutyric acid (GABA), myelin, phospholipids, ethanolamine glycerophospholipid, ethanolamine plasmalogen, galactosylceramide

Lipid abnormalities in succinate semialdehyde dehydrogenase () deficient mouse brain provide additional evidence for myelin alterations

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INTRODUCTION

Heritable succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (Aldh5a1) deficiency (OMIM 271980, 610045) is an inborn error of metabolism disrupting the final step in GABA catabolism [START_REF] Gibson | Murine succinate semialdehyde dehydrogenase (SSADH) deficiency, a heritable disorder of GABA metabolism with epileptic phenotype[END_REF]. The autosomal-recessively inherited defect leads to significant increases in GABA and γ-hydroxybutyric acid (GHB) in body fluids derived from affected patients. Elevated GHB in urine often results in the primary route of identification (via routine organic acid analysis), and represents the metabolic basis for the eponym, γhydroxybutyric aciduria [START_REF] Gibson | Disorders of beta-and gamma-amino acids in free and peptide-linked forms[END_REF]. Clinically, the phenotype is that of non-specific mild to severe neurological involvement, including psychomotor retardation, hypotonia, ataxia, reduced to absent speech development, and seizures in some patients [START_REF] Pearl | Inherited disorders of neurotransmitters in children and adults[END_REF]. Treatment options are limited, and have been predominantly symptomatic, such as carbamazepine for seizures, haloperidol for psychiatric manifestations, and rytalin for attention-deficit disorder [START_REF] Gibson | 4-Hydroxybutyric acid and the clinical phenotype of succinic semialdehyde dehydrogenase deficiency, an inborn error of GABA metabolism[END_REF]. The most widely employed pharmacotherapy is Sabril R (vigabatrin; γ-vinyl GABA), an inhibitor of the enzymatic reaction preceding SSADH, GABA transaminase. The rationale for vigabatrin intervention derives from its capacity to decrease the production of GHB (Fig. 1) [START_REF] Gibson | Vigabatrin therapy in six patients with succinic semialdehyde dehydrogenase deficiency[END_REF]. Nonetheless, since vigabatrin also elevates cerebral GABA [START_REF] Gibson | Vigabatrin therapy in six patients with succinic semialdehyde dehydrogenase deficiency[END_REF], its utility in SSADH deficiency may require reevalauation since this is already a hyperGABAergic syndrome.

The absence of effective therapies, coupled to inadequate insights into underlying pathophysiology, led Hogema and colleagues [START_REF] Hogema | Pharmacologic rescue of lethal seizures in mice deficient in succinate semialdehyde dehydrogenase[END_REF] to develop a knockout model for this disorder (Aldh5a1 -/-mice). These animals are born at the expected Mendelian frequencies (~25%), fail to gain measurable weight, transition from absence seizures at ~ 2 weeks of life to generalized tonic-clonic convulsion at 3-4 weeks of life and eventual lethal status-epilepticus thereafter [START_REF] Cortez | Absence seizures in succinic semialdehyde dehydrogenase deficient mice: a model of juvenile absence epilepsy[END_REF][START_REF] Buzzi | Succinic semialdehyde dehydrogenase deficiency: GABA(B) receptor-mediated function[END_REF][START_REF] Wu | Status epilepticus in mice deficient for succinate semialdehyde dehydrogenase: GABAA receptor-mediated mechanisms[END_REF]. A variety of metabolic disturbances have been detected in Aldh5a1 -/-mice in both physiological fluids and tissue extracts. These include significant changes in GABA, GHB,

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4 homocarnosine (the GABA-histidine dipeptide), succinate semialdehyde, 4,5dihydroxyhexanoate, D-2-hydroxyglutarate, guanidinoacetate, guanidinobutyrate, glutamine, neuroactive steroids, monamines and others [START_REF] Gupta | Therapeutic intervention in mice deficient for succinate semialdehyde dehydrogenase (Gamma-hydroxybutyric aciduria)[END_REF][START_REF] Gupta | Murine succinate semialdehyde dehydrogenase deficiency[END_REF][START_REF] Gupta | Seizure evolution and amino acid imbalances in murine succinate semialdehyde dehydrogenase (SSADH) deficiency[END_REF][START_REF] Gibson | Oxidant stress and neurodegeneration in murine succinic semialdehyde dehydrogenase (SSADH) deficiency, In: Diseases of Neurotransmission-from bench to bed[END_REF][START_REF] Jansen | Increased guanidino species in murine and human succinate semialdehyde dehydrogenase (SSADH) deficiency[END_REF][START_REF] Gibson | Focal neurotransmitter alterations in mice deficient for succinate semialdehyde dehydrogenase[END_REF][START_REF] Struys | Metabolism of γ-hydroxybutyrate to D-2hydroxyglutarate in mammals: further evidence for D-2-hydroxyglutarate transhydrogenase[END_REF]. Alterations in GABA B -and GABA Areceptors levels and isoforms, in the presence of unchanged glutamatergic transmission, suggest that imbalanced inhibitory/excitatory neurotransmission may underlie the mechanism of seizure transition [START_REF] Buzzi | Succinic semialdehyde dehydrogenase deficiency: GABA(B) receptor-mediated function[END_REF][START_REF] Wu | Status epilepticus in mice deficient for succinate semialdehyde dehydrogenase: GABAA receptor-mediated mechanisms[END_REF]. Early lethality in Aldh5a1 -/-mice afforded the opportunity to examine new treatment modalities, with preliminary studies revealing that antagonists of both the GHB-and GABA B -receptors, as well as application of the non-physiological amino acid taurine, led to significant lifespan extension for these mice [START_REF] Gupta | Therapeutic intervention in mice deficient for succinate semialdehyde dehydrogenase (Gamma-hydroxybutyric aciduria)[END_REF].

Earlier studies from our laboratory utilized microarray gene expression studies in an attempt to identify novel pharmacotherapeutic targets for Aldh5a1 -/-mice [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF]. Unexpectedly, those investigations led to the discovery of significant alterations in myelin-associated genes in the hippocampus and cortex of these animals. To further dissect these myelin abnormalities, we have performed a comprehensive examination of brain phospholipid mass and composition in Aldh5a1 -/-and Aldh5a1 +/+ mice, accompanied by analysis of individual phospholipid fatty acid composition. Hexose-ceramides, (e.g., glucosyl-and galactosyleceramides, markers of myelin integrity) were also quantified in the same tissues. Our data suggest that fatty acid anomalies in glycerophospholipid fractions may play a pathophysiological role in this disorder.

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5

METHODS

Animals

Age-and sex-matched wild-type (Aldh5a1 +/+ ) and gene-ablated mice (Aldh5a1 For the majority of studies, 5-7 animals each were utilized for analyses, with the exception of hexose ceramide studies which employed n=4 animals for each genotype.

Thin layer chromatography

Individual phospholipid classes and neutral lipids were separated by thin layer chromatography. Whatman silica gel-60 plates (20 x 20 cm, 250 µm) were heat-activated at 110°C for 1 hour and samples streaked onto plates. Phospholipids were separated using chloroform:methanol:acetic acid:water (55:37.5:3:2 by volume [START_REF] Jolly | Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation[END_REF]. Lipid fractions were identified using authentic standards (Doosan-Serday, Englewood Cliffs, NJ and NuChek Prep, Elysian, MN). Phospholipid mass was determined by assaying for lipid phosphorus content of individual separated lipid classes separated [START_REF] Rouser | Quantitative analysis of phospholipids by thin layer chromatography and phosphorus analysis of spots[END_REF]. Neutral lipids were separated in petroleum ether:diethyl ether:acetic acid (75:25:1.3 by vol) [START_REF] Marcheselli | Quantitative analysis of acyl group composition of brain phospholipids, neutral lipids, and free fatty acids, in Neuromethods 7 Lipids and Related Compounds[END_REF]. Cholesterol was assayed using an iron binding assay after separation by TLC as described previously [START_REF] Hara | Lipid extraction of tissues with a low-toxicity solvent[END_REF].

Fatty acid analysis

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The ethanolamine glycerophospholipids (EtnGpl), choline glycerophospholipids (ChoGpl), phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) fractions were separated by high performance liquid chromatography [START_REF] Dugan | Separation of phospholipids by high pressure liquid chromatography: all major classes including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine[END_REF]. Each fraction was subjected to basecatalyzed transesterification, converting the phospholipid acyl chains to fatty acid methyl esters (FAME). To each fraction, 2 mL of 0.5 M KOH dissolved in anhydrous methanol were added [START_REF] Brockerhoff | Determination of the positional distribution of fatty acids in glycerolipids[END_REF]. FAME were extracted from the methanol using 2 mL of n-hexane and the n-hexane phase containing the FAME was removed. The lower phase was re-extracted two more times with 3 mL of n-hexane and these washes were combined with the original aliquot.

Individual fatty acids were separated by gas liquid chromatography (GLC) and quantified using flame ionization detection using an SP-2330 column (0.32 mm ID x 30 m length) and a

Trace GLC (ThermoElectron, Austin, TX) equipped with dual autosamplers and dual flame ionization detectors (FID). For each fatty acid, peak areas were converted to moles using a standard curve from commercially purchased standards (NuChek Prep, Elysian, MN) and 17:0 as the internal standard.

Hexose-Ceramide Determinations

Tandem mass spectrometry, using multiple reaction monitoring with isotope dilution, was employed for determination of glycosphingolipids, which included sulphatides as well as galactosylceramide and glucosylceramide. The isoforms of hexose ceramide (glucosyl-and galactosylceramide are not differentiated) and sphingomyelin were quantified, and the ratio (hexose-ceramide/sphingomyelin) determined to correct for individual variation. Complete methodological details have been previously reported [START_REF] Mills | Synthesis of novel internal standards for the quantitative determination of plasma ceramide trihexoside in Fabry disease by tandem mass spectrometry[END_REF][START_REF] Mills | Monitoring the clinical and biochemical response to enzyme replacement therapy in three children with Fabry disease[END_REF][START_REF] Mills | Measurment of urinary CDH and CTH by tandem mass spectrometry in patients hemizygous and heterozygous for Fabry disease[END_REF].

Statistics

Statistical analysis was performed using Instat 2 from GraphPad (San Diego, CA).

Statistical significance was assessed using two-way, unpaired Student's t-test, with p<0.05 considered significant.

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7

RESULTS

Brain Phospholipid Mass and Composition

The total phospholipid mass was not altered in Aldh5a1 -/-mice; however, EtnGpl mass was decreased 20% in these animals (Table 1). This result is consistent with the 30 % decrease in ethanolamine plasmalogen mass previously reported [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF]. Thus, Aldh5a1gene ablation in the mouse affected only EtnGpl, and more specifically the ethanolamine plasmalogen subclass.

Brain Individual Phospholipid Fatty Acid Composition

The fatty acid composition of the four main brain phospholipids, EtnGpl, choline glycerophospholipid (ChoGpl), phosphatidylserine (PtdSer) and phosphatidylinositol (PtdIns), was analyzed (Tables 2345). In EtnGpl, the proportion of 18:0 was increased 1.05-fold, while proportions of 20:1n-9 and 22:4n-6 were decreased 37% and 10%, respectively. Although the total n-3 and n-6 fatty acid contents were not statistically different, the n-3 to n-6 ratio was increased 1.1-fold. In ChoGpl, proportions of 22:6n-3 were increased 1.2-fold in Aldh5a1 -/- mice, accounting for a significant increase in the n-3 to n-6. In PtdIns, the proportion of 20:4n-6

was decreased 15% affecting the n-3 to n-6 ratio which was increased 1.7-fold, indicating a net increase in n-3 fatty acids relative to n-6. In PtdSer, proportions of 18:1n-9 and 20:1n-9 decreased 20% and 37% respectively, while the proportion of 22:6n-3 was increased 1.1-fold.

The increase in n-3 fatty acids was accompanied by decreased n-6 fatty acids, thereby affecting the n-3 to n-6 ratio.

Brain Neutral Lipid Mass

Because brain is highly enriched in cholesterol, the effects of Aldh5a1 deletion on brain cholesterol mass were determined (Table 6). Aldh5a1 -/-mice did not have a significant change in brain cholesterol mass as compared to wild-type. Because neither the brain total phospholipid content nor the brain cholesterol content was change, there was no change in the cholesterol to phospholipid ratio.

Brain Glycosphingolipids

Glucosyl-and galactosylceramides (e.g., hexose-ceramides) are major components of myelin in both grey and white matter. Accordingly, these were quantified in extracts of brains derived from Aldh5a1 -/-and Aldh5a1 +/+ mice (Fig. 2). The ratio of hexose-ceramide to sphingomyelin was employed to correct for variations between individual animals (n=4 animals each genotype: Aldh5a1 +/+ , 17.9 + 4.2; Aldh5a1 -/-, 10.5 + 6.0; unpaired one-tailed t-test, p=0.0449). Although we predicted a decrease, ganglioside analysis in the same brain fractions did not reveal significant alterations between genotypes (data not shown).

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9

DISCUSSION

In the current study, we observed a significant decrease in ethanolamine glycerophospholipid mass that was consistent with the 30 % decrease in ethanolamine plasmalogen reported by Donarum and coworkers [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF]. The data of Table I indicate that the loss of Aldh5a1 activity in the mouse affected only the ethanolamine glycerophospholipid fraction of brain, and more specifically the ethanolamine plasmalogen subclass. Ethanolamine plasmalogen is a major constituent of myelin, and certain species of this plasmalogen are unique to the myelin sheath [START_REF] Hack | The diminution of the myelin ethanolamine plasmalogen in brain of the Jimpy mouse and brain and spinal cord of the Quaking mouse as visualized by thin-layer chromatography[END_REF]. Isolated decreases in ethanolamine plasmalogen fractions have been detected in other mouse models and human disorders. Ethanolamine plasmalogen was significantly decreased in brain and spinal cord derived from the Quaking mouse, as well as brain derived from the Jimpy mouse [START_REF] Hack | The diminution of the myelin ethanolamine plasmalogen in brain of the Jimpy mouse and brain and spinal cord of the Quaking mouse as visualized by thin-layer chromatography[END_REF]. The Quaking mouse has been extensively studied in relation to its neurological phenotype, characterized as severe tremor of voluntary movements with onset at ~ day of life 10. In this animal, an 1 Mb deletion in the qk gene leads to significant oligodendrocyte alterations and CNS dysmyelination [START_REF] Lorenzetti | Deletion of the Parkin coregulated gene causes sterility in the quaking viable mouse mutant[END_REF]. The Jimpy mouse harbors an Xlinked mutation in proteolipid protein (PLP) which results in severe CNS dysmyelination affecting glial and neural populations [START_REF] Harsan | New insights on neuronal alterations in Jimpy mutant brain[END_REF]. PLP is a key protein required for maintenance of the multilamellar myelin sheath structure and axonal integrity [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF], and its decreased expression in Aldh5a1 -/-brain was demonstrated by both microarray and quantitative RTPCR [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF]; Western blotting of PLP is in progress to support these initial findings. These observations suggest that myelin abnormalities occurring in the Aldh5a1 -/-brain may best be characterized as dysmyelination, although to date there has been no convincing evidence for dysmyelination in patients with SSADH (Aldh5a1) deficiency.

Abnormalities of ethanolamine glycerophospholipids have been observed in human neurological disorders and induced-states in rodents. Decreased ethanolamine glycerophosphatides are detected in gray matter and frontal lobe myelin obtained post-mortem

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10 from a 57 year-old patient with Refsum's disease [START_REF] Macbrinn | Lipid composition of the nervous system in Refsum's disease[END_REF]. In white matter myelin derived from 13/17 patients with multiple sclerosis (MS), the ratio of different molecular species of enthanolamine plasmalogen was comparable to control values but was significantly altered in the remaining 4 MS cases and in samples derived from a patient with subacute sclerosing panencephalitis [START_REF] Boggs | Comparison of two molecular species of ethanolamine plasmalogen in multiple sclerosis and normal myelin[END_REF]. Sun and coworkers [START_REF] Sun | Effects of chronic ethanol administration on rat brain phospholipids metabolism[END_REF] demonstrated adaptive increases in ethanolamine plasmalogen synthesis in synaptosomes derived from rats treated chronically with ethanol.

Conversely, Bichenkov and Ellingson [START_REF] Bichenkov | Effects of transient ethanol exposure on the incorporation of [(3)H]ethanolamine into plasmalogen in the differentiating CG-4 oligodendrocyte cell line[END_REF] demonstated that ethanolamine plasmalogen synthesis in the CG-4 oligodendrocyte cell line could be both up-and down-regulated dependent upon the developmental time period of acute ethanol exposure. Since ethanol and GHB have many similar pharmacological properties (and synergistic capabilities) [START_REF] Wong | Gamma-hydroxybutyric acid: neurobiology and toxicology of a recreational drug[END_REF], it is interesting to speculate that the ethanolamine plasmalogen abnormalities detected in Aldh5a1 -/-mice correlate with chronic GHB exposure in the CNS, which is a testable hypothesis in rodent model systems treated chronically with GHB.

Galactosylceramide (hexose-ceramide; galactosecerebroside) represents the major sphingolipid of the myelin membrane [START_REF] Zoller | Oligodendrocyte-specific ceramide galactosyltransferase (CGT) expression phenotypically rescues CGT-deficient mice and demonstrates that CGT activity does not limit brain galactosylceramide level[END_REF][START_REF] Bradl | Myelin dysfunction/degradation in the central nervous system: why are myelin sheaths susceptible to damage?[END_REF]. In the current study, galactosylceramide was notably lower in Aldh5a1 -/-mice in comparison to wild-type littermates. Quantitation of galactosylceramide is generally considered a sensitive marker for myelin destruction [START_REF] Thuillier | Immunological determination of galactosylceramide level in blood as a serum index of active demyelination[END_REF]. The ratio of hexose-ceramide to sphingomyelin represents a reliable indicator of whether the hexoseceramide is actually lower in a particular study group. In our investigation, sphingomyelin was employed as an indicator of overall cellular glycolipid content in lieu of going through more sophisticated assays for absolute galactosylceramide quantitation. It is important to note that sphingomyelin mass was not altered in these mice (Table I). Vanier [START_REF] Vanier | Lipid changes in Niemann-Pick disease type C brain: personal experience and review of the literature[END_REF] noted a severe loss of galactosylceramide in Niemann-Pick type C patients exhibiting the severe or late-infantile forms of the disease. Similarly, Hara and Taketomi [START_REF] Hara | Cerebral lipid and protein abnormalities in Menkes' steely-hair disease[END_REF] analyzed cerebral lipids in an infant male with Menkes' disease, noting a significant decrease in galactosylceramide in myelin sheath associated

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11 with defective copper metabolism. Mice unable to produce galactosylceramide exhibit thin, unstable myelin which often leads to progressive demyelination and significant motor coordination defects [START_REF] Zoller | Oligodendrocyte-specific ceramide galactosyltransferase (CGT) expression phenotypically rescues CGT-deficient mice and demonstrates that CGT activity does not limit brain galactosylceramide level[END_REF][START_REF] Esch | Sphingolipid profile in the CNS of the twitcher (globoid cell leukodystrophy) mouse: a lipidomics approach[END_REF][START_REF] Marcus | Sulfatide is essential for the maintenance of CNS myelin and axon structure[END_REF]. For example, in the twitcher mouse (a model of globoid cell leukodystrophy) there is a reduction of C24:1 and C24:1 hydroxylated galactosylceramide species, associated with the acute demylination in this animal model [START_REF] Esch | Sphingolipid profile in the CNS of the twitcher (globoid cell leukodystrophy) mouse: a lipidomics approach[END_REF]. We hypothesize that the reduced galactosylceramide detected in our studies (Fig. 2) correlates with the reduced myelin sheath thickness quantified in cortical sections of Aldh5a1 -/-mice [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF].

To extend our findings of decreased ethanolamine glycerophospholipid level in the brain of Aldh5a1 -/-mice, we subjected each of the individual phospholipid fractions to exhaustive fatty acid analyses (Tables 2345). There were significant differences for Aldh5a1 -/-mice in comparison to wild-type littermates for a variety of fatty acids; however, the most consistent results were increased n-3 (omega-3) fatty acids, principally 22:6 n-3 (docosahexaenoic acid; DHA). We predict that the alterations detected in omega-3 fatty acids and plasmalogens in the Aldh5a1 -/- mice can be tracked to the endoplasmic reticulum and specific alterations in selected desaturase enzymes, a hypothesis that is currently under investigation in the laboratory. The phosphatidylserine fraction derived from Aldh5a1 -/-mice showed a significant decrease in monounsaturated fatty acids coupled with a corresponding increased in polyunsaturated fatty acids (Table 5). The resultant ratios of unsaturated to saturated fatty acids were also altered.

Similar alterations in unsaturated fatty acids have been detected in cerebral myelin and synaptosomes from phenylketonuric brain and hyperphenylalaninemic chick embryos [START_REF] Johnson | Fatty acid composition of lipids in cerebral myelin and synaptosomes in phenylketonuria and Down syndrome[END_REF][START_REF] Alejandre | Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos[END_REF],

as well as in cerebral lipids derived from an infantile case of Menkes' disease [START_REF] Hara | Cerebral lipid and protein abnormalities in Menkes' steely-hair disease[END_REF]; however, it is likely that the mechanisms leading to unsaturated fatty acid anomalies in these disorders are quite different from those in the Aldh5a1 animal model.. plasmalogen abnormalities in Aldh5a1 -/-mice, it is of interest that the white matter pool of plasmalogens turns over much more slowly than pools associated with grey matter, suggesting that grey matter pools have a signaling role. The processes at play in Aldh5a1 deficiency, however, may have quite different etiologies while simultaneously contributing to the phenotype.

Correlation of clinical and biochemical features between

For example, increased signal intensity in the basal ganglia may derive from chronic oxidative damage, and recent data has shown that GHB ellicits oxidant stress [START_REF] Sgaravatti | Gamma-hydroxybutyric acid induces oxidative stress in cerebral cortex of young rats[END_REF]. GABA is key to the distribution of myelin sheaths in early development [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF], and almost certainly plays a role (with GHB) in alterations of inhibitory neurotransmission that have been detected in Aldh5a1 -/-mice that underlie the genesis of seizures [START_REF] Buzzi | Succinic semialdehyde dehydrogenase deficiency: GABA(B) receptor-mediated function[END_REF][START_REF] Wu | Status epilepticus in mice deficient for succinate semialdehyde dehydrogenase: GABAA receptor-mediated mechanisms[END_REF]. Accordingly, the alterations we have seen in myelin (and likely fatty acid profiles) are almost certainly a secondary phenomenon related to primary accumulation of GHB and GABA in brain.

One limitation in the current investigation is that our analyses were focused on a single developmental time point (17-19 days of life), a period of rapid myelination in certain regions of the CNS [START_REF] Donarum | Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency[END_REF]. Further studies assessing myelin and lipid structures in younger Aldh5a1 -/-mice, as well as those who have survived the early-onset seizure activity associated with this disorder, are planned. These studies are currently in progress. Values are expressed as mass (nmol/g ww) and represent mean ± SD, n = 5-7. The * indicates a significant difference between groups, p<0.05.

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12 (

 12 Aldh5a1 deficient patients and knockout mice remains challenging. Both patients and mice manifest a neurological phenotype A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT ataxia in mice and patients, hypotonia and retardation in patients), but a vexing issue is the occurrence of seizures, which is uniform in the mouse model yet only ~ 50% prevalent in human patients. Neuroimaging in patients indicates signal abnormalities in deep grey matter structures (basal ganglia), yet myelin anomalies would indicate white matter alterations. With regard to

Fig. 1 .

 1 Fig. 1. Interactions of the Krebs cycle and GABA shunt in central nervous system. The site of the block in patients with SSADH deficiency (and Aldh5a1 -/-mice) is depicted by the italicized enzyme name Aldh5a1. Abbreviations employed: α-KG, α-ketoglutarate; glu, glutamate; GHB, γ-hydroxybutyrate; GABA, γ-aminobutyrate; SSA, succinate semialdehyde. Increased intermediates detected in Aldh5a1 -/-mice and SSADH-deficient patients are shown with upward-

Fig. 2 .

 2 Fig.2. Ratio of hexose-ceramide (including glucosyl-and galactosylceramide) to sphingomyelin in brain tissue derived from wild-type (Aldh5a1 +/+ ) and mutant (Aldh5a1 -/-) mice as determined

Table 1 . Brain phospholipid mass in Aldh5a1 +/+ and Aldh5a1 -/-mice

 1

		Aldh5a1 +/+	Aldh5a1 -/-	Aldh5a1 +/+	Aldh5a1 -/-
			nmol/g ww				mole%
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
	Ptd 2 Gro	1051	142	922	332	2.3	0.3	2.0	0.5
	PtdGro	959	175	899	260	2.1	0.5	2.0	0.5
	PtdOH	464	128	554	249	0.9	0.2	1.2	0.4
	EtnGpl	11852	1025	9539	1587*	24.1	1.5	21.6	2.1*
	PtdIns	2228	217	1952	402	4.7	0.5	4.4	0.4
	PtdSer	4207	852	4134	1155	8.6	1.5	9.2	1.1
	ChoGpl	23126	2004	21883	3447	48.3	2.0	49.5	2.3
	CerPCho	4357	863	4482	1126	9.0	1.9	10.0	1.0
	Total	48244	3559	44366	7986
	Values are expressed as mole percentage and represent mean ± SD, n = 5-7. Abbreviations:
	Ptd 2 Gro: cardiolipin; PtdGro: phosphatidylglycerol; PtdOH: phosphatidic acid; EtnGpl:
	ethanolamine glycerophospholipids; PtdIns: phosphatidylinositol; PtdSer: phosphatidylserine;
	ChoGpl: choline glycerophospholipid; CerPCho: sphingomyelin. The * indicates a significant
	difference between groups, p<0.05.

Table 2 . Fatty acid composition of brain ethanolamine glycerophospholipids from Aldh5a1 +/+ and Aldh5a1 -/-mice

 2

	EtnGpl
	mole %	Aldh5a1 +/+	Aldh5a1 -/-
	FAME	Mean	SD
	16:0	8.6	0.9	8.5	1.1
	18:0	26.1	0.8	27.4	0.3*
	18:1 n-9	8.4	1.2	7.6	0.4
	18:1 n-7	1.7	0.2	1.5	0.0
	18:2 n-6	0.7	0.1	0.8	0.2
	20:1 n-9	0.8	0.3	0.5	0.05*
	20:3 n-6	0.7	0.2	0.6	0.1
	20:4 n-6	23.4	0.6	22.9	0.4
	22:4 n-6	5.9	0.3	5.3	0.2*
	22:6 n-3	23.7	1.0	25.0	1.0
	sat	34.8	1.3	35.9	1.4
	unsat	65.2	1.3	64.1	1.4
	MUFA	10.9	1.6	9.6	0.4
	PUFA	54.4	1.0	54.6	1.3
	n-3	23.7	1.0	25.0	1.0
	n-6	30.7	0.6	29.6	0.5
	n-3/n6	0.77	0.04	0.84	0.03*
	unsat/sat	1.88	0.10	1.79	0.11
	PUFA/MUFA	5.13	0.90	5.71	0.28
	MUFA/sat	0.31	0.06	0.27	0.02
	PUFA/sat	1.57	0.06	1.52	0.09
		n=7		n=5
	Values are expressed as mole percentage and represent mean ± SD, n = 5-7. The * indicates a
	significant difference between groups, p<0.05. Abbreviations employed for Tables 2-6: FAME,
	fatty acid methyl esters; n-3, n-6, n-7, n-9, etc-refers to omega-3, omega-6, etc; sat, saturated;
	unsat, unsaturated; MUFA, monounsaturated fatty acids; PUFA, polyunsatured fatty acids; chol,
	cholesterol; PL, phospholipids

Table 3 . Fatty acid composition of brain choline glycerophospholipids from Aldh5a1 +/+ and Aldh5a1 -/-mice

 3

	ChoGpl
	mole %	Aldh5a1 +/+	Aldh5a1 -/-
	FAME	Mean	SD
	16:0	51.2	1.3	51.5	0.7
	16:1	0.9	0.1	0.9	0.1
	18:0	10.7	0.5	10.6	0.3
	18:1 n-9	17.1	0.2	16.7	0.1*
	18:1 n-7	5.1	0.2	5.0	0.1
	18:2 n-6	1.3	0.5	1.2	0.1
	20:1 n-9	0.6	0.1	0.6	0.1
	20:3 n-6	0.4	0.0	0.5	0.1*
	20:4 n-6	9.0	0.6	8.5	0.3
	22:4 n-6	0.6	0.1	0.7	0.1
	22:6 n-3	3.3	0.4	3.9	0.3*
	sat	61.9	1.0	62.1	0.6
	unsat	38.1	1.0	37.9	0.6
	MUFA	23.6	0.3	23.2	0.1
	PUFA	14.4	1.0	14.6	0.4
	n-3	3.3	0.4	3.9	0.3*
	n-6	11.1	0.9	10.8	0.2
	n-3/n6	0.30	0.05	0.36	0.03*
	unsat/sat	0.62	0.03	0.61	0.01
	PUFA/MUFA	0.61	0.05	0.63	0.02
	MUFA/sat	0.38	0.01	0.37	0.01
	PUFA/sat	0.23	0.02	0.24	0.01
	Values are expressed as mole percentage and represent mean ± SD, n = 5-7. The * indicates a
	significant difference between groups, p<0.05.

Table 4 . Fatty acid composition of brain phosphatidylinositol from Aldh5a1 +/+ and Aldh5a1 - /-mice

 4

	PtdIns
	mole %	Aldh5a1 +/+	Aldh5a1 -/-
	FAME	Mean	SD
	16:0	12.2	1.7	13.9	2.8
	18:0	37.5	1.4	37.9	4.1
	18:1 n-9	6.2	0.5	6.0	0.2
	18:1 n-7	2.9	0.5	2.5	0.4
	20:4 n-6	34.9	2.2	29.5	4.0*
	22:4 n-6	1.9	0.6	2.1	0.7
	22:6 n-3	3.4	0.8	4.2	0.5
	sat	49.7	1.5	51.9	4.7
	unsat	50.3	1.5	48.1	4.7
	MUFA	10.2	2.5	8.6	0.6
	PUFA	40.2	3.1	37.9	5.2
	n-3	3.4	0.8	4.8	1.2
	n-6	36.8	2.7	31.7	4.2*
	n-3/n6	0.09	0.02	0.15	0.04*
	unsat/sat	1.01	0.06	0.94	0.16
	PUFA/MUFA	4.15	0.96	4.58	0.64
	MUFA/sat	0.20	0.05	0.20	0.08
	PUFA/sat	0.81	0.08	0.74	0.15
	Values are expressed as mole percentage and represent mean ± SD, n = 5-7. The * indicates a
	significant difference between groups, p<0.05.

Table 5 . Fatty acid composition of brain phosphatidylserine from Aldh5a1 +/+ and Aldh5a1 -/- mice

 5 Values are expressed as mole percentage and represent mean ± SD, n = 5-7. The * indicates a significant difference between groups, p<0.05.

	PtdSer
	mole %	Aldh5a1 +/+	Aldh5a1 -/-
	FAME	Mean	SD
	16:0	3.8	0.3	3.7	0.4
	18:0	42.1	0.6	41.9	0.9
	18:1 n-9	10.1	1.3	8.0	0.5*
	18:1 n-7	1.3	0.1	1.1	0.0
	18:2 n-6	0.5	0.1	0.4	0.1
	20:1 n-9	0.8	0.2	0.5	0.04*
	20:3 n-6	0.8	0.3	0.7	0.2
	20:4 n-6	5.8	0.4	5.3	0.3
	22:4 n-6	4.7	0.3	4.8	0.3
	22:6 n-3	30.1	1.2	32.9	0.8*
	sat	46.0	0.6	45.6	1.0
	unsat	54.0	0.6	54.4	1.0
	MUFA	12.2	1.6	9.6	0.5*
	PUFA	41.8	1.4	44.8	0.7*
	n-3	30.1	1.2	32.9	0.8*
	n-6	11.7	0.6	11.8	1.3
	n-3/n6	2.59	0.16	2.82	0.34
	unsat/sat	1.18	0.03	1.19	0.05
	PUFA/MUFA	3.49	0.64	4.65	0.22*
	MUFA/sat	0.27	0.04	0.21	0.01*
	PUFA/sat	0.91	0.03	0.98	0.04*

Table 6 . Total brain cholesterol and phospholipid mass in Aldh5a1 +/+ and Aldh5a1 -/-mice

 6

		Aldh5a1 +/+	Aldh5a1 +/+	Aldh5a1 -/-	Aldh5a1 -/-
	nmol/gww	Mean	SD	Mean	SD
	Chol	23870	4024	20495	2844
	Total PL	48244	3559	44366	7986
	Chol/PL ratio	0.50	0.10	0.47	0.06

ACKNOWLEDGEMENTS

The authors are indebted to Carole L. Haselton for excellent technical assistance in lipid analyses. Supported in part by NIH NS 40270 (KMG) and IP20-RR17699-01 (EJM).


M Newman

I F Musgrave
email: ian.musgrave@adelaide.edu.au

M Lardelli
email: michael.lardelli@adelaide.edu.au

Please cite this article as: M. Newman, I.F. Musgrave, M. Lardelli, Corrigendum to "Alzheimer disease: Amyloidogenesis, the presenilins and animal models" [Biochem. Biophys. Acta 1772 (2007) 285-297], BBA -Molecular Basis of Disease (2007), doi: 10.1016/j.bbadis.2006.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.