Miguel Agustin Contreras

William Louis Ries

Srinivasan Shanmugarajan

Gonzalo Arboleda
email: gharboledab@unal.edu.co

Inderjit Singh

M.D. Avtar Kaur Singh
email: hhuuavtar.singh@med.va.govu

Shanmugara- Jan Srinivasan

Factors that affect postnatal bone growth retardation in the twitcher murine

Keywords: ceramide, GH: growth hormone, IGF-1: insulin-like growth factor-1, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, qPCR: quantitative polymerase Galactosylsphingosine, Insulin-like growth factor-1, Krabbe disease, lysosomal disorders, osteopenia, psychosine, twitcher mice

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A C C E P T E D

M A N U S C R I P T

Introduction

The infantile form of Krabbe disease (Globoid cell leukodystrophy) is the most common, a severe, rapidly progressing, neurodegenerative and invariably fatal, inherited metabolic disease produced by mutations on the β-galactosylcerebrosidase gene, which expresses a lysosomal enzyme that participates in the degradation of several glycosphingolipids [START_REF] Wenger | Galactosylceramide Lipidosis: Globoid cell Leukodystrophy (Krabbe Disease)[END_REF][START_REF] Kolodny | Globoid leukodystrophy[END_REF]. As a consequence, galactosylsphingosine (psychosine, also a substrate of the enzyme [START_REF] Igisu | Progressive accumulation of toxic metabolite in a genetic leukodystrophy[END_REF]) accumulates in the nervous system [START_REF] Igisu | Progressive accumulation of toxic metabolite in a genetic leukodystrophy[END_REF] and peripheral organs [START_REF] Kobayashi | Globoid cell leukodystrophy is a generalized galactosylsphingosine (psychosine) storage disease[END_REF]. The availability of a mutant mouse (twitcher mouse; twi), that is a morphologically, genetically and enzymatically authentic murine model of the human disease [START_REF] Suzuki | The twitcher mouse. A model of human globoid cell leukodystrophy (krabbe's disease)[END_REF], has been a valuable tool to advance our understanding of the development/progression of the disease in the central nervous system. In the nervous system, accumulation of psychosine exerts a cytotoxic effect especially on myelin-producing cells (oligodendrocytes) [START_REF] Tanaka | Effects of psychosine (galactosylsphingosine) on the survival and the fine structure of cultured Schwann cells[END_REF],

with further induction of myelin degeneration, astrocytic gliosis, axonal loss and presence of multinucleate giant (globoid) cells [START_REF] Wenger | Galactosylceramide Lipidosis: Globoid cell Leukodystrophy (Krabbe Disease)[END_REF]. Psychosine-induced local inflammatory response in the nervous system (induction of proinflammatory cytokines and intermediates) [START_REF] Ohno | MHC class II antigen expression and T-cell infiltration in the demyelinating CNS and PNS of the twitcher mouse[END_REF][START_REF] Levine | IL-6 and TNFalpha expression in brains of twitcher, quaking and normal mice[END_REF][START_REF] Taniike | Expression of major histocompatibility complex class I antigens in the demyelinating twitcher CNS and PNS[END_REF][START_REF] Itoh | Immunohistological study of globoid cell leukodystrophy[END_REF] plays a role in the early apoptotic loss of oligodendrocytes [START_REF] Haq | Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line[END_REF][START_REF] Zaka | Psychosine-induced apoptosis in a mouse oligodendrocyte progenitor cell line is mediated by caspase activation[END_REF] observed in Krabbe patients [START_REF] Giri | Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease[END_REF][START_REF] Jatana | Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine[END_REF] and twitcher mice [START_REF] Taniike | An apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell leukodystrophy[END_REF] brains.

Twitcher mouse develops clinical signs by day 20th, characterized by stunted growth, twitching and hind leg weakness; and the disease progresses to a near-terminal stage by day 40-45 [START_REF] Wenger | Galactosylceramide Lipidosis: Globoid cell Leukodystrophy (Krabbe Disease)[END_REF]. The twitching sign is associated with the loss of oligodendrocytes [START_REF] Matsushima | Absence of MHC class II molecules reduces CNS demyelination, microglial/macrophage infiltration, and twitching in murine globoid cell leukodystrophy[END_REF][START_REF] Tanaka | The twitcher mouse: accumulation of galactosylsphingosine and pathology of the sciatic nerve[END_REF][START_REF] Jacobs | The pathogenesis of globoid cell leucodystrophy in peripheral nerve of the mouse mutant twitcher[END_REF]; however, the cause of decreased somatic growth is unclear. Multiple etiologies could underlie this growth defect; but alteration of hypothalamic-hypophysial axis

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

function may play a causative role. Growth hormone (GH), secreted by the anterior pituitary into the circulation, has long been recognized as one of the principal factors that control postnatal growth [START_REF] Kaplan | Hormonal regulation of growth and metabolic effects of growth hormone[END_REF]. GH acts as a promoter of skeletal long bone growth and development as well as chondrocyte maturation [START_REF] Isaksson | Growth hormone stimulates longitudinal bone growth directly[END_REF][START_REF] Reinecke | Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo[END_REF], by receptor mediated induction of the transcription of genes whose expressed proteins are important in mediating its diverse effects on body growth and metabolism [START_REF] Carter-Su | Molecular mechanism of growth hormone action[END_REF]. GH also stimulates the expression of hepatic and extrahepatic insulin-like growth factor-1 (IGF-1), that plays an essential GH-mediated anabolic role on skeletal and non skeletal tissues [START_REF] Mathews | Regulation of insulin-like growth factor I gene expression by growth hormone[END_REF][START_REF] Baker | Role of insulin-like growth factors in embryonic and postnatal growth[END_REF][START_REF] Zapf | Insuline-like factor I actions on somatic growth[END_REF][START_REF] Schlechter | Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin[END_REF][START_REF] Roberts | Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver[END_REF][START_REF] Roith | The somatomedin hypothesis: 2001[END_REF].

We have previously reported that psychosine accumulation mediates the induction of cytokines in twi liver, thus compromising some of its functions [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF]. In the present report, , we studied the presence of endogenous (psychosine; Sph-1P) and humoral (IGF-1) factors that may be responsible for the stunted growth of femora of twi mice, to help identify possible mechanisms that may be playing a role in the postnatal somatic growth retardation of twi mouse. Our data indicate that: i) psychosine accumulates in femora of twi mice; ii) osteoclast function of twi femora (bone resorption) is enhanced; iii) tartrate resistant acid phosphatase (TRAP) activity is increased in the growth plate; iv) twi mouse femora present alteration of sphingolipid homeostasis, without signs of change in Sph-1P or a significant decrease in sphingomyelin or an increase in ceramide pool; v) liver secreted IGF-1 level is drastically reduced in twi mice plasma, and vi) IGF-1 transcripts and protein levels are significantly down regulated in twi liver. This study reports for the first time evidence that psychosine, acting either endogenously in the bone (local accumulation) or in conjunction with a disrupted endocrinal signal (IGF-1), are

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

potential factors that mediate the alteration in bone growth, and hence the somatic growth of twi mice.

Material and methods

Animal model

All animal work for this study was performed under a protocol approved by the Institutional Animal Care and Use Committee. Twitcher heterozygote breeding pairs (C57BL/6J twi +/-) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and twi (-/-) (twi) and age-matched littermates wild type wt (+/+) (wt) were used for this study at postnatal day 33-34. Wt and twi animals were sacrificed with an overdose of anesthetic and tissues were removed, frozen in liquid nitrogen and stored at -70 o C.

Femora analysis of psychosine and sphingolipid levels by HPLC-mass spectrometry

For lipidomic analysis, bones (left and right femur) were defrosted in ice-cold phosphate buffered saline and the muscle tissue removed manually. Bones were weighed, enveloped in aluminum foil, frozen in liquid nitrogen and crushed in a mortar.

Quantitative high pressure liquid chromatography-tandem mass spectrometry analysis of psychosine and bioactive sphingolipids were performed on organics solvent extracts derived from wt and twi crushed bones, at the University Lipidomics Core facility [START_REF] Bielawski | Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatographytandem mass spectrometry[END_REF].

Calcein bone labeling

To define abnormal bone growth in the twi mouse, a modified histomorphometric analysis was performed using double labeling with calcein as a marker [START_REF] Ono | Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts[END_REF] and dehydrated with serial changes in 70-100% ethanol. The specimens were embedded in methylmethacrylate and serial 4-6 µm sections were stained by Goldner's trichrome method for light microscopy analysis [START_REF] Horn | A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail[END_REF] or stained for TRAP activity using a histochemical kit (Sigma-Aldrich) as described elsewhere [START_REF] Shanmugarajan | Transgenic mice with OIP-1/hSca overexpression targeted to the osteoclast lineage develop an osteopetrosis bone phenotype[END_REF].

Plasma and liver analysis of Insulin-like Growth Factor-1

Blood from wt and twi mice was collected in EDTA-K containing vaccutainer tubes (BD Biosciences, Franklin Lakes, NJ), and the plasma was separated by centrifugation at 900xg for 10min (Allegra X-15R centrifuge, Beckman-Coulter,

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

Fullerton, CA) and stored at -70 o C. Levels of IGF-1 were determined using an ELISA kit (Quantikine, mouse IGF-1 immunoassay according to manufacturer instructions (R&D Systems, Minneapolis, MN).

2.6. Bone analysis of cytokines (Tumor Necrosis Factor-alpha and Interleukin-6):

Expression of cytokines (TNF-α and IL-6) was detected by ELISA in the supernatant of lysis buffer bones' extracts (RIPA buffer, Thermo Scientific Inc., Waltham, MA). Bones were individually processed as indicated for the mass spectrometric analysis and extracted with 120 uL of lysis buffer. ELISA analysis was performed according to manufactured instructions (TNF-α: eBioscience, San Diego, CA;

IL-6: BD Biosciences, San Jose, CA) using 50 uL of bone's extract. Plates' readings were performed in a Spectra Max 190 ELISA reader (Molecular Devices, Sunnyvale, CA).

Quantitative polymerase chain reaction

Liver total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) and the purified RNA used for the synthesis of cDNA. Single-strand cDNA was synthesized from 2µg of total RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc, Hercules, CA) according to manufacturer's instructions. qPCR was conducted using iCycler (iCycler iQ, Bio-Rad), specific primers for mouse IGF-1 (Cat. number PPM03387E; SABiosciences, Frederick, MD) and iQ SYBR Green Supermix (Bio-Rad).

Thermal cycling conditions were as follows: activation of DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30s and 60°C for 30s. The expressions of target genes were normalized with respect to the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All samples were run in

A C C E P T E D M A N U S C R I P T duplicate.
Statistical analysis was performed by student-t test (Graph Path InStat3, GraphPath Software Inc., San Diego, CA) and the difference at p <0.05 was considered statistically significant.

Results

Retarded growth of long bones (femora) of Twitcher mouse and Galactosylsphingosine (psychosine) accumulation

Postnatal growth curves show that twi mouse body weight is severely inhibited by day 20 (Fig. 1A), as reported by several laboratories [START_REF] Luzi | Generation of a mouse with low galactocerebrosidase activity by gene targeting: a new model of globoid cell leukodystrophy (Krabbe disease)[END_REF][START_REF] Gasperi | Transgenic rescue of Krabbe disease in the twitcher mouse[END_REF][START_REF] Galbiati | Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease[END_REF]. Taking long bones (femora) as representatives of the animals' growth, we analyzed their physical differences between wt and twi mice. The analysis of femora of wt and twi at post natal day 32-33 indicated that twi bones have smaller size (Fig. 1B) and weighed 27% less than wt mice bones (Fig. 1C). Furthermore, determination of psychosine levels by mass spectrometry indicated that psychosine accumulates in twi bones of adults animals (33 days old), at levels 14 fold higher than the levels found in wt (Table 1).

Histomorphologic analysis of Twitcher femora

Examination of stained wt and twi femora under light microscopy revealed differences in cortical bone thickness, cellular organization (isogenous groups) and width of the growth plate, between wt and twi animals (Fig. 2A,B). The metaphyseal region of twi bones also showed marked structural differences, with reduction of trabecular bone (stained in green) mainly in the region of the secondary spongiosa (Fig. 2C,D). Overall,

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

there was a marked decrease in cortical bone thickness and the number of trabeculae, indicating the twi mouse shows an osteopenic phenotype.

Bone growth/remodeling functions (osteoblastic deposition and osteoclastic resorption) in femora of wild type and twitcher mice

To gain insight into the possible cause of reduced bone in twi femora, the boneforming capacity of osteoblasts was examined by the deposition of calcein. The calcein labeling along the cortical bone (seen as two layers of florescent green lines under the fluorescence microscopic analysis) showed no difference between wt and twi (Fig. 3A,B), indicating no differences in the mineral apposition rate. However, there was a substantial reduction of bone deposition (green fluorescence) in the metaphyseal and epiphyseal region of twi femora (Fig. 3A,B). To evaluate whether enhanced osteoclastic activity is the cause of the observed reduced calcein deposition in both regions, we performed histochemical analysis for tartrate-resistant acid phosphatase (TRAP) activity.

TRAP-positive osteoclasts were identified along the base of the growth plate and most of the trabeculae and on the endosteal surface of the diaphysis in both wt and twi mice bone showed activity (Fig. 4A,B). However, TRAP staining was stronger in twi bone, and included the narrow growth plate of the twi femur. These data indicate that psychosineinduced alterations in twi bone preferentially affects the function of osteoclasts and suggests that enhanced osteoclastic resorption may be playing a role in the bone growth retardation observed in twi animals. To evaluate possible changes in the osteoclast-osteoblast coupling factor sphingosine-1 phosphate (Sph-1P) [START_REF] Ryu | Sphingosine 1phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling[END_REF] and to better understand the cause of reduced cellularity reported in twi bone marrow [START_REF] Galbiati | Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease[END_REF], we studied the levels of sphingolipid in femora. The analysis of the wt and twi femora by mass spectrometry indicated a significant increase (1.3 fold) in the total sphingomyelin (SM) pool found in the twi (341 + 10 nmoles/g) when compared to wt bones (263 + 11 nmoles/g; p < 0.0001)(Fig. 5A).

A C C E P T E

The observed increase is mainly a contribution of the saturated species C16:0-SM (1. 2). On the other hand, the analysis of the ceramide (CM) pool did not indicate a significant difference between wt and twi bones (Fig. 5B).

However, significant changes were observed in the individual saturated species C20:0-CM (0.7 fold) and C22:0-CM (1.4 fold), DHC16:0 (0.8 fold), Sph (1.4 fold), and the unsaturated species C24:1-CM (1.3 fold) (Table 2). Interestingly, the most abundant species (C24:1) in SM as well as in CM was significantly elevated in both pools; and the dihydro-C16:0-CM (DHC16:0; a de novo CM synthesis indicator [START_REF] Spiegel | Sphingosine-1-phosphate: an enigmatic signalling lipid[END_REF]) was significantly decreased. Taking into account that the changes observed in the total SM pool and SM species represent an increase in concentrations in twi as compared with wt, and minor variations in some CM saturated and unsaturated species, these data suggest that psychosine accumulation in twi bones is interfering with SM and CM turnover.

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

To rule out a possible psychosine-induced inflammatory process as demonstrated in the brains and livers of twitcher mice [START_REF] Levine | IL-6 and TNFalpha expression in brains of twitcher, quaking and normal mice[END_REF][START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF], we determined the bone's levels of the cytokines TNF-α and IL-6 (Table 4). Because the cytokine analysis did not indicate a significant difference between wild type and twitcher mice (Table 4), the results suggest that psychosine is not exerting its effect by inducing the expression of mediators of inflammation.

3.5 Insulin-like growth factor-1 (IGF-1) levels in plasma and mRNA expression and protein levels in liver IGF-1 is a GH-dependent growth factor produced mainly by the liver, and it is known to regulate growth and bone remodeling [START_REF] Elefteriou | Regulation of bone remodeling by the central and peripheral nervous system[END_REF]. ELISA analysis of plasma levels of IGF-1 in wt and twi animals indicated that IGF-1 levels were significantly reduced by 37.6% in twi animals as compared to wt (234.4 + 26.4 vs 148.7 + 20.5 ng/ml in wt and twi respectively; p < 0.002)(Fig. 6), and decreases as the disease progresses (Fig. S1A,B). Because plasma circulating IGF-1 is of hepatic origin and liver is also affected by psychosine induced-disease in twi mice [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF], we investigated the mRNA expression and protein levels in liver of wt and twi mice. The hepatic expression of IGF-1 mRNA was down regulated by 59.3% in adult animals (1.9 + 0.4 in wt vs. 0.8 + 0.3 folds in twi; p < 0.005)(Fig. 7A), whereas IGF-1 protein levels were decreased by 60.4% (1.7 + 0.2 in wt vs. 0.7 + 0.3 ng/mg of protein in twi; p < 0.0001)(Fig. 7B) in twi liver as compared with wt. This result indicated a strong alteration of the liver functions in twi animals.

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

Discussion

Twitcher mouse is an animal model of the genetic disorder Globoid cell leukodystrophy (Krabbe disease) [START_REF] Suzuki | The twitcher mouse. A model of human globoid cell leukodystrophy (krabbe's disease)[END_REF][START_REF] Duchen | Hereditary leucodystrophy in the mouse: the new mutant twitcher[END_REF][START_REF] Kobayashi | The Twitcher mouse: an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease)[END_REF]. These animals develop clinical signs by day 20 (stunted growth, twitching, hind leg weakness), and they reach a near-terminal stage by day 40-45 [START_REF] Wenger | Galactosylceramide Lipidosis: Globoid cell Leukodystrophy (Krabbe Disease)[END_REF]. While the twitching sign is associated with oligodendrocytes (myelinforming cell) loss [START_REF] Matsushima | Absence of MHC class II molecules reduces CNS demyelination, microglial/macrophage infiltration, and twitching in murine globoid cell leukodystrophy[END_REF][START_REF] Tanaka | The twitcher mouse: accumulation of galactosylsphingosine and pathology of the sciatic nerve[END_REF][START_REF] Jacobs | The pathogenesis of globoid cell leucodystrophy in peripheral nerve of the mouse mutant twitcher[END_REF], the cause of twi mouse decreased growth is unclear. It is well established that psychosine accumulates in nervous tissue [START_REF] Igisu | Progressive accumulation of toxic metabolite in a genetic leukodystrophy[END_REF], and exerts an inflammatory response characterized by induction of proinflammatory cytokines, inducible nitric oxide synthase, oxidative imbalance and early oligodendrocyte cell death [START_REF] Levine | IL-6 and TNFalpha expression in brains of twitcher, quaking and normal mice[END_REF][START_REF] Haq | Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line[END_REF][START_REF] Zaka | Psychosine-induced apoptosis in a mouse oligodendrocyte progenitor cell line is mediated by caspase activation[END_REF][START_REF] Giri | Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease[END_REF][START_REF] Jatana | Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine[END_REF][START_REF] Taniike | An apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell leukodystrophy[END_REF][START_REF] Khan | Peroxisomal participation in psychosine-mediated toxicity: Implications for Krabbe's disease[END_REF]. As the disease pathology progresses, the inflammatory process compromises further brain homeostasis, altering subcellular functions of peroxisomes [START_REF] Khan | Peroxisomal participation in psychosine-mediated toxicity: Implications for Krabbe's disease[END_REF][START_REF] Haq | Dysfunction of peroxisomes in twitcher mice brain: a possible mechanism of psychosine-induced disease[END_REF] and possibly the mitochondria [START_REF] Strasberg | Cerebrosides and psychosine disrupt mitochondrial functions[END_REF][START_REF] Igisu | Inhibition of cytochrome c oxidase by psychosine (galactosylsphingosine)[END_REF]. In parallel with the central nervous system events, psychosine also accumulates in non-nervous tissues/organs, affecting their homeostasis [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF][START_REF] Galbiati | Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease[END_REF]. Our data demonstrated that psychosine also accumulates in long bones (femora) of twitcher mice, where it may exert cytotoxic effects, thus supporting the notion that globoid cell leukodystrophy (Krabbe disease) is a generalized storage disease [START_REF] Kobayashi | Globoid cell leukodystrophy is a generalized galactosylsphingosine (psychosine) storage disease[END_REF], and adding another tissue to the list of those that accumulate psychosine [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF][START_REF] Galbiati | Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease[END_REF][START_REF] Nozawa | Novel procedure for measuring psychosine derivatives by an HPLC method[END_REF][START_REF] Whitfield | Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry[END_REF][START_REF] Ida | An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher)[END_REF]. Such accumulation, however, does not compare to the levels found in twi liver or brain (3 and 27 folds higher than in bone, respectively) [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF].

Many local [START_REF] Ryu | Sphingosine 1phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling[END_REF][START_REF] Karsdal | Osteoclasts secrete non-bone derived signals that induce bone formation[END_REF][START_REF] Boyce | Functions of RANKL/RANK/OPG in bone modeling and remodeling[END_REF][START_REF] Matsuo | Osteoclast-osteoblast communication[END_REF], humoral/endocrine [START_REF] Isaksson | Growth hormone stimulates longitudinal bone growth directly[END_REF][START_REF] Reinecke | Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo[END_REF][START_REF] Schlechter | Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin[END_REF] and central and peripheral nervous system-derived factors [START_REF] Elefteriou | Regulation of bone remodeling by the central and peripheral nervous system[END_REF][START_REF] Singh | Cocaine and amphetamine-regulated transcript may regulate bone remodeling as a circulating molecule[END_REF] regulate the functions of the specialized cells such as osteoblasts (bone deposition) and osteoclasts (bone resorption) responsible for In most tissues, SM degradation (hence increase in CM levels) is associated with activation of stress response/cell death [START_REF] Hannun | Functions of ceramide in coordinating cellular responses to stress[END_REF], and Sph-1P increase is associated with antiapoptotic/cell survival signal [START_REF] Cuvillier | Suppression of ceramide-mediated programmed cell death by sphingosine-1phosphate[END_REF][START_REF] Spiegel | Sphingosine-1-phosphate in cell growth and cell death[END_REF]. In addition, in bone Sph-1P is a factor that regulates the osteoclast-osteoblast coupling during bone growth/remodeling [START_REF] Ryu | Sphingosine 1phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling[END_REF][START_REF] Matsuo | Osteoclast-osteoblast communication[END_REF]. In general, our sphingolipid analysis resembles the changes reported for twi liver [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF]; with insignificant changes in known apoptotic (C16:0-CM) or antiapoptotic (Sph-1P)

sphingolipid fractions [START_REF] Spiegel | Sphingosine-1-phosphate: an enigmatic signalling lipid[END_REF][START_REF] Osawa | Roles for C16-ceramide and sphingosine 1-phosphate in regulating hepatocyte apoptosis in response to tumor necrosis factor-alpha[END_REF]. However, an increase was found in total SM fraction, and in most of the SM and CM containing saturated-unsaturated fatty acids, including the most abundant species (C24:1) in SM and in CM. On the other hand, the dehydro-C16:0 (DHC16:0; an indicator of de novo CM synthesis [START_REF] Spiegel | Sphingosine-1-phosphate: an enigmatic signalling lipid[END_REF]) was significantly decreased and Sph accumulated in twi mice. These data suggest that psychosine accumulation may interfere with SM degradation and CM synthesis in twi bones. Furthermore, the lipidomic analysis showing no significant changes on Sph-1P between wt and twi mice bone rules out that this sphingolipid may be playing a role in the osteoclast-osteoblast uncoupling [START_REF] Ryu | Sphingosine 1phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling[END_REF]. These results suggest that the levels of psychosine may not reach a threshold that is enough to trigger stress response/cell death in twi bones. Consistent with these results, analysis of the inflammation marker TNF-α and IL-6 were found not to be altered, which strongly suggests that the psychosine-induced pathology in twitcher bones is not dependent or potentiated by the induction of mediators of inflammation, as demonstrated in the brain and liver of those animals [START_REF] Levine | IL-6 and TNFalpha expression in brains of twitcher, quaking and normal mice[END_REF][START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF] or studies on cell culture [START_REF] Giri | Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease[END_REF]. On the other hand, psychosine accumulation may be interfering with cell signaling at the level of the receptor sites (lipids rafts), as suggested recently [START_REF] White | Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture[END_REF]. Future studies should be aimed to identify the cellular distribution of the accumulated psychosine in long bones.

Liver is the main organ that contributes to the blood circulating IGF-1 pool [START_REF] Roberts | Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver[END_REF][START_REF] Roith | The somatomedin hypothesis: 2001[END_REF][START_REF] Ohlsson | The dual effect theory[END_REF]; with IGF-1 playing an important role in bone physiology [START_REF] Zapf | Insuline-like factor I actions on somatic growth[END_REF][START_REF] Ernst | Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I[END_REF][START_REF] Hock | Insulin-like growth factor I has independent effects on bone matrix formation and cell replication[END_REF]. Taking into consideration that liver functions are compromised by psychosine-induced inflammation in twi animals [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF], it was not surprising that mRNA expression and protein levels of IGF-1 were found to be down regulated in the liver of twi mice, supporting the drastic decrease in IGF-1 plasma levels observed in those mice. These results indicate an additional psychosine-mediated metabolic dysfunction in twi liver (synthesis of IGF-1), and suggest that reduction in IGF-1 plasma levels may be an additional factor contributing to the observed impaired balance (uncoupling) between osteoclast-osteoblast function, and hence to the bone growth stunting in twi mice. Studies performed in double gene disrupted mice (liver IGF-1-deficient and acid labile subunit (ALS) knockout) support our results [START_REF] Yakar | Circulating levels of IGF-1 directly regulate bone growth and density[END_REF]; dramatic decrease in IGF-1 levels in both animals is consistent with decreased bone growth. Indeed, the fewer trabeculae in the metaphysis is a sign of osteopenia; and the smaller growth plate in both cases is consistent with the narrowing of the growth plate observed with aging, which is associated with decrease in circulating and local production of IGF-1 [START_REF] Rosen | Clinical review 123: Anabolic therapy for osteoporosis[END_REF][START_REF] Rosen | Insulin-like growth factors and bone: the osteoporosis connection[END_REF][START_REF] Rosen | Association between serum insulin growth factor-I (IGF-I) and a simple sequence repeat in IGF-I gene: implications for genetic studies of bone mineral density[END_REF]. Furthermore, additional factors such as the endogenous presence of cytokines (as seen in brain and liver) [START_REF] Levine | IL-6 and TNFalpha expression in brains of twitcher, quaking and normal mice[END_REF][START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF] or alterations in the hypothalamic-pituitary axis in the nervous system [START_REF] Hegedus | Neurofibromin regulates somatic growth through the hypothalamic-pituitary axis[END_REF], may also play a role in the derangements of bone growth. Recent studies have reported drastic changes in twi mice bone marrow functions [START_REF] Galbiati | Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease[END_REF].

Our data and that of others demonstrate the complexity ofthe development and progression of the psychosine-induced pathology in the nervous system [11-13, 36, 43] and in peripheral organs [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF][START_REF] Galbiati | Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease[END_REF], and highlight the importance of peripheral organs for the overall organism homeostasis [START_REF] Contreras | Psychosine-induced alterations in peroxisomes of twitcher mouse liver[END_REF]. Based on these observations, we hypothesize that progression of inflammation in the twi central nervous system could affect hormone secretion which may play a role in the homeostasis of peripheral organs, such as in bone growth regulation [START_REF] Scarborough | Cytokine modulation of pituitary hormone secretion[END_REF][START_REF] O'connor | Regulation of IGF-I function by proinflammatory cytokines: at the interface of immunology and endocrinology[END_REF].

In summary, we have identified two potential factors that may be affecting bone growth in twi mice. We report that psychosine accumulates in bones, where either maybe by itself as an endogenous factor or in conjunction with a decrease of plasma IGF-1 levels, preferentially impaired osteoclastic function, and hence stunted bone growth.

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

Therefore, recent therapeutic treatments [START_REF] Escolar | Transplantation of umbilical-cord blood in babies with infantile Krabbe's disease[END_REF][START_REF] Cartier | Hematopoietic stem cell gene therapy in Hurler syndrome, globoid cell leukodystrophy, metachromatic leukodystrophy and Xadrenoleukodystrophy[END_REF] might consider the supplementation/induction of endocrinous/paracrine factors (i.e., GH-IGF-1).

Combination therapy may prove to be of benefit for the central nervous system [START_REF] Frago | Growth hormone (GH) and GH-releasing peptide-6 increase brain insulin-like growth factor-I expression and activate intracellular signaling pathways involved in neuroprotection[END_REF][START_REF] Aberg | Aspects of growth hormone and insulin-like growth factor-I related to neuroprotection, regeneration, and functional plasticity in the adult brain[END_REF][START_REF] Zaka | Insulin-like growth factor-1 provides protection against psychosine-induced apoptosis in cultured mouse oligodendrocyte progenitor cells using primarily the PI3K/Akt pathway[END_REF] and peripheral organs, and ultimately for the amelioration of the development and progression of the disease pathology in twi mice and Krabbe patients.

10 3. 4 .

 104 D M A N U S C R I P T ACCEPTED MANUSCRIPT Sphingolipid homeostasis and cytokine markers in femora of twitcher mouse

  3 fold), C18:0-SM (1.2 fold), C20:0-SM (1.4 fold), C22:0-SM (1.4 fold), and C24:0-SM (1.2 fold); and of the unsaturated species C22:1-SM (1.4 fold), C24:1-SM (1.4 fold) and C26:1-SM (1.1 fold) (Table

A

  C C E P T E D M A N U S C R I P Tbone growth/remodeling[START_REF] Boyce | Functions of RANKL/RANK/OPG in bone modeling and remodeling[END_REF]. Morphologic and functional analysis of wt and twi femora demonstrated reduced content of cortical and trabecular bone indicating that the twi mouse shows an osteopenic phenotype, possibly a consequence of enhanced osteoclastic bone resorption (TRAP activity) without much alteration of osteoblastic function (calcein deposition). These data indicate that psychosine-induced alterations in twi bone may uncouple bone deposition from bone resorption. It affects preferentially osteoclastic function and suggests that over time an enhanced osteoclastic resorption may be playing a role in the premature closure of the growth plate; and hence is a contributing factor to the reduction in bone growth in twi animals. Interestingly, in another animal model of lysosomal disease, mucopolyssacharidosis type VII (deficiency of the enzyme βglucuronidase), there is metabolite accumulation mediated bone alteration by impairment of both osteoclastic and osteoblastic functions[START_REF] Monroy | Abnormal osteoclast morphology and bone remodeling in a murine model of a lysosomal storage disease[END_REF].

Figure 1 .

 1 Figures captions:

Figure 2 .

 2 Figure 2. Morphologic differences between the femur from wild type and twitcher mice.

Figure 3 .

 3 Figure 3. Bone deposition (osteoblasts activity) and resorption (osteoclasts activity)

Figure 4 .

 4 Figure 4. Sphingolipid concentration in wild type and twitcher femora. The levels of

Figure 5 .

 5 Figure 5. Insulin-like growth factor-1 levels in the plasma of twitcher mice. Plasma

Figure 6 .

 6 Figure 6. Expression and protein level of insulin-like growth factor-1 in twitcher mice

  : Samples represent Mean U+ U SD of psychosine concentrations found in femora from wild type (n = 3 sets) and twitcher (n = 4 sets) mice, determined at postnatal day 32-33. (Set includes the left and the right femur from each animal). **: p < 0.004 A C C E P T E D M A N U S C R I P T

  ( a ): Individual sphyngomyelin fractions were determined by mass spectrometry in defleshed bone (wet weight) organic extracts. Values represent the average ± SD for n = 5 sets per group. p <: Statistical significance as indicated, versus wild type; ns: not significant.A C C E P T E DM A N U S C R I P T

  ( a ): Samples represent Mean U+U SD of cytokines concentrations (tumor necrosis factoralpha, TNF-α; Interleukin-6, IL-6) found in femora from wild type (n = 4 sets) and twitcher (n = 5 sets) mice, determined at postnatal day 33-35. (Set includes the left and the right femur from each animal).

Table 1 :

 1 Level of galactosylsphingosine (psychosine) in wild type and twitcher mice femora

Table 2 :

 2 Determination of the sphingomyelin fractions composition in wild type and twitcher femora

			Sphingomyelin fractions a
				(nmoles/g tissue)
		C14:0	C16:0 C18:0 C20:0	C22:0 C24:0	C26:0
	C18:1	C20:1	C22:1	C24:1	C26:1
	Wild type 1.07 + 0.05 110 + 4 39 + 3 4.7 + 0.5 14 + 2 36 + 2 0.35 + 0.05
	2.9 + 0.3 0.36 + 0.07 2.6 + 0.4 52 + 5 0.75 + 0.04
	Twitcher 1.23 + 0.17 143 + 6 48 + 2 6.5 + 0.6 19 + 2 42 + 3 0.33 + 0.05
	3.2 + 0.3 0.44 + 0.06 3.6 + 0.4 73 + 4 0.85 + 0.05
	p <		ns	0.0001 0.001	0.001	0.002 0.005	ns
	ns	ns	0.005 0.0002	0.01

Table 4 :

 4 Level of cytokines (TNF-α and IL-6) in wild type and twitcher mice femora

		TNF-α (pmoles / g tissue) a IL-6
	Wild type	86 + 56	152 + 79
	Twitcher	112 + 76	149 + 63

ACCEPTED MANUSCRIPT

Acknowledgements

The authors would like to thank Ms. Joyce Bryan, Dr. Narender Nath and the members of the Lipidomics Core Facility of the Medical University of South Carolina for the excellent technical assistance. This work was supported by Grants from the Extramural Research Facilities Program of the National Center for Research Resources (C06 RR018823 and C06 RR015455) and from the National Institutes of Health (NS-22576, NS-34741, NS-37766, NS-40810 and AG-25307).

were determined by ELISA assay (B), as described in Materials and Methods. Graph represents the average ± SD for the number of animals indicated per group. Analysis was performed in mice 33-34 days old. **: p < 0.005 (n = 3); ***: p < 0.0001 (n = 6).

Asterisks denote significant statistical differences from wild type littermates. WT: wild type mice; TW: Twitcher mice.


Marcin Dampc

Ewelina Szymańska

Mariusz Brygida Mielewska

Ewelina Szyma Ska

Mariusz Zubek

Ionization and ionic fragmentation of tetrahydrofuran molecules by electron collisions

Keywords:

HAL is

Introduction

The interaction of high energy radiation, electrons, ions and radicals with biomolecules, in particular with the DNA fragments, nucleic purine and pyrimidine bases, phosphate units and deoxyribose sugar, has been extensively studied to uncover effects of primary and secondary particles in radiation damage of living cells and other biological systems [1][2][3][4][5][6]. The secondary electrons, which are produced in large quantities by the primary ionizing particles in the biological material, may cause single-and double strand breaks in the polynucleotides of DNA through site selective cleavage of the molecular bonds. The molecular bonds in the repeated sugar-phosphate backbones and also in the four fundamental nucleic bases attached to the backbone by covalent bonds are ruptured efficiently through processes of dissociative electron attachment, neutral fragments dissociation and dissociative ionization. For example, the gas phase studies of dissociative electron attachment to purine (adenine and guanine) and pyrimidine (thymine and cytosine) bases showed that they undergo electron-induced fragmentation in the subionization energy range (<10 eV) where the density of the secondary electrons is high [7,8]. These secondary electrons produced by the primary radiation, which in the pre-thermalization period possess energies greater than 10 eV but below 100 eV, will efficiently induce dissociative ionization of the targets, because of the high associated value of collision cross section for low electron impact energies. In order to determine the most sensitive part of the DNA or RNA molecular chains to electron-induced bond rupture thorough studies of reactions to electrons of their building structures and combinations of these structures, e. g. nucleic bases and phosphate-sugar groups were investigated [9][10][11].

These recent studies demonstrated that it is the deoxyribose (2-deoxy-D-ribose), which is most likely damaged in producing strand breaks in DNA or RNA [12,13].

Deoxyribose, C 5 H 10 O 4 (D) is a constituent of the sugar-phosphate backbone of the DNA and RNA polynucleotide chains and bonds with the nucleic bases. In the chain, it is in the furanose form of a five-membered carbon, oxygen ring with the CH 2 group attached to C (2) atom and providing bonds to phosphate group and nuclei bases at C(3) and C(5), respectively (figure 1a). However, in the gas phase D is predominantly in the pyranose form of sixmembered ring and in order to study specifically the strength of the furanose ring under electron interactions several analogues, -tetrahydrofurfuryl alcohol (THFA), 3hydroxytetrahydrofuran (3HTHF) and tetrahydrofuran (THF) molecules have been used as corresponding targets [14]. The THF, C 4 H 8 O molecule (figure 1b) is often considered as the simplest analogue to deoxyribose. In the two other analogues, as compared to THF, the hydrogen atoms at position ( 2) and ( 3) are replaced by CH 2 OH and OH radicals, respectively. Thus, THF molecules became recently a frequent subject of experimental investigations of electron interactions, elastic scattering [15][16][17], vibrational excitation [17][18][19] and dissociative electron attachment [20,21] in the gas phase. Vibrational excitation [18] and H -desorption [22] in the electron interaction with deposited thin film layers were also studied.

In this paper we present investigations of the electron-induced ionization and ionic fragmentation of the THF molecules in the gas phase, in the energy range from the ionization threshold up to 150 eV. The cation mass spectra at selected electron energies and the cation fragmentation efficiencies curves were measured using technique of mass spectrometry. A significant amount of work were also put into determination of appearance energies (AE) of ionic fragments. Earlier, electron impact mass spectra of the THF molecule were measured by Gallegos and Kiser [23] at 70 eV and by Collin and Conde-Caprace [24] in the 10-50 eV range (see this reference for other earlier works). These authors also determined the AE of the most abundant ionic fragments and analyzed possible paths of the THF fragmentation from their thermochemical calculations. The results of Ref.s [23,24] are displayed in the NIST database [START_REF]NIST chemistry web book[END_REF]. Duffield et al. [START_REF] Duffield | [END_REF] obtained mass spectra of five-membered heterocycles including the THF and studied fragmentation modes of the THF by deuterium labeling method. Very recently, Fuss et al. [27] measured the time-of-flight ionic mass spectra of the THF over extended energy range 50-5000 eV, however with a limited mass resolution. These authors also carried out the total ion collection measurements to determine total ionization cross section of the THF. The total ionization cross section of the THF was calculated by Mo ejko and Sanche [28] in the binary-encounter-Bethe approach. Photon induced ionic fragmentation of the THF in the gas phase was studied very recently by Mayer et al [29] using tunable synchrotron radiation. The AEs obtained for the observed ionic molecular fragments are lower (by 1-3 eV) than those obtained by electron impact in Ref.s [23,24]. Applied threshold photoelectron photoion coincidence technique allowed breakdown curves for the detected ions to be measured in the energy range up to 35eV.

The measurements of this work were performed using a quadrupole mass spectrometer for the ion mass analysis and the mass spectra were recorded at selected electron energies. A number of well resolved mass peaks were detected in the mass range 10-72 amu and were assigned to corresponding ionic fragments. These spectra resolved some inconsistencies between earlier works (NIST data) and mass spectra published very recently [27], as will be described later. The AE of the identified cations were determined from the ion efficiency curves measured over the threshold regions by applying a fitting procedure. The cation efficiency curves measured in the 5-150 eV energy range were normalized with reference to mass spectra to determine contributions of the individual cations to the total ionization cross section. Motivation of the present work is to provide data of the partial and total ionization efficiencies which may serve as the input parameters in modeling radiation-induced damage in the biomolecular systems.

Experimental

The measurements of the cation mass spectra at a fixed electron energy and the ionic fragment efficiencies of the THF as a function of electron energy were carried out using quadrupole mass spectrometer EPIC 300 (Hiden Analytical Ltd.). The spectrometer incorporates an electron ionizer, an ion extractor and focusing lens electrodes, a quadrupole mass filter and a secondary electron multiplier for ion detection. The THF vapour was delivered into the system by a narrow stainless steel capillary positioned 5 mm from the ionization cage. The spectrometer was mounted in a vacuum chamber evacuated by a 60 ls -1 turbomolecular pumping unit to a base pressure of 2 × 10 -7 mbar which increased to the operating pressure of 1 × 10 -6 mbar when introducing the target THF vapour. During the measurements electron incident current in the ionizer was 2-10 µA. Linear dependences between the operating pressure, the incident electron current and the detected ion signal were maintained. As the THF cations formed in the ionic fragmentation may carry initial translation energy, it was also ensured that the measured mass spectra are independent of the spectrometer tuning for ion kinetic energies in the range 0-3 eV.

The estimated transit time from the ionization region to the detector of 50 amu ions, at 3 eV ion kinetic energy was 50 µs. The ionic fragment efficiency curves obtained in the 5-150 eV range were corrected for the energy variation of the electron incident beam. Here, argon Ar + ions yields were recorded in a mixture with the THF and compared with argon ionization cross section [30] to deduce the correction factor. The background signal was measured at the base pressure of 2 × 10 -7 mbar and subsequently subtracted from the original spectra.

The mass dependence of transmission of the quadrupole spectrometer was tested by measuring the mass spectra of carbon dioxide and comparing these results with the literature cross sections for CO 2 fragment ions [31]. It was found that the transmission is constant in the mass region from the 28 amu (CO + /CO 2 ) to 44 amu (CO 2 + /CO 2 ), which is the region in which the majority of the THF cation fragments appear. In determination of the relative cation efficiencies of the THF it was assumed that the ion transmission does not vary appreciably for masses outside of this region and that the transmission for masses of 15, 71 and 72 amu is equal to that of the 28-44 amu region. This assumption may introduce increased uncertainty in the relative intensities of the above ions. However, it was found that when varying their contributions into the total ion efficiency curve it had small effect on the overall shape of the curve. The electron energy was calibrated against known AE of Ar + (15.75 eV) to within ± 0.15 eV. The measurements of Ar + yield in the region above AE enabled a value of 600 meV for the energy spread of the incident electron beam to be deduced.

The anhydrous liquid THF was placed in a stainless steel container attached to a gas line.

which was maintained at the room temperature. Heating of the sample was not required as the THF has sufficiently high vapour pressure at this temperature. It was degassed several times under low pressure to remove contaminating gases, nitrogen, oxygen and water vapour, before introducing into the spectrometer by a leak valve, which was heated to avoid THF condensation.

The THF mass spectra measured simultaneously showed decreasing intensities of these contaminations to a level much lower than that of the THF ions. THF was purchased from Sigma-Aldrich Chemie with a stated purity of 99.9 %.

The AEs of the observed ionic molecular fragments were determined from the ion efficiency yields measured in an energy region of about 5 eV positioned symmetrically against the expected AE threshold. To determine accurate values of AE the efficiency curves were fitted with an exponential near-threshold cross section function

σ(E) = 0 , E E A (1) σ(E) = c(E -E A ) n , E > E A
which was convoluted with the Gaussian electron energy spread. The parameters involved in the fitting are, E A the appearance energy, c the scaling factor and n the exponential constant. The above procedure based on an exponential expression (1) was introduced by Märk [32] and further developed by Matt et al [33] and used extensively thereafter [14]. The uncertainties of the obtained AEs which take into account the uncertainty in energy calibration and the standard deviations of the mean values of AEs are given in table 2 together with the values of AEs.

Results and discussion

Electron collisions with THF molecules in the gas phase lead to ionization into the parent Figure 3 shows ionization efficiency curves obtained in the threshold regions for masses of 71, 42, 41, 29 amu and illustrates the high quality of the fitting obtained in the determination of the ionic AEs from the indicated exponential functions.

C 4 H 8 O + cation

Mass spectrum of THF

The cation peaks in the mass spectrum of THF appear in two distinct groups occurring in the mass ranges 26-31 and 37-45 amu, respectively. Two further intense peaks are seen at masses 71 and 72 amu. Low intensity peaks are detected at mass 15 amu and also at 1 and 2 amu (not shown in figure 2). The most abundant cation is observed at mass 42 amu and the second most abundant cation at mass 41 amu, both having intensities higher than that of the parent C 4 H 8 O + ion of mass 72 amu. The inset of figure 2 shows the mass spectrum in the range 46-62 amu, which covers the region of low abundance cations with relative intensities below 0.6 %. The 54, 55 and 57 amu peaks may correspond to impurities, because their intensities decreased with time of the measurements. The spectrum of figure 2 is in very good agreement with the NIST database [START_REF]NIST chemistry web book[END_REF].

The time-of-flight spectrum of Fuss et al [27] measured at 1000 eV shows unresolved groups of cation peaks near 29, 40 and 72 amu, consistent with our high resolution mass spectrum of figure 2. It also shows a wider mass peak of high intensity around 52 amu that is not confirmed by our results (see inset of figure 2), and neither by the NIST data. This disagreement cannot be caused by the difference in electron energies of both measurements, as the 52 amu peak in the Fuss et al data appears also and dominates at lower energies. For example, at 150 eV it gives a 42 % contribution to the total ionization cross section. The above disagreement between both mass spectra could be rationalized if the 52 amu ions have a relatively short lifetime and decay in-flight before being detected in our mass spectrometer. However, our estimations of the ion transit times based on the experimental details in both measurements show that the times are comparable (30-50 µs).

Some conclusions may be drawn from comparison of the THF mass spectrum of the present work and that of the other analogues, THFA and 3HTHF [14] with the cation mass spectrum of D [12]. The mass spectra of all four molecules display groups of cations in the 26-32 and 39-45 amu regions. The 15-20 amu cations in THFA and 3HTHF have lower relative intensities than that in D while in THF that group is reduced to a single peak at 15 amu. The group of 55-61 amu cations are seen in the D and 3HTHF spectra but not in that of THFA and THF. None of the cation groups around 70 amu in the mass spectra of the analogues resemble that of the D spectrum, which contains more fragments than the analogue molecules. The parent ion has the highest abundance in the case of THF (32%) and it is reduced to 10.8 % in 3HTHF [41]. In THFA the parent ion abundance is lower than 1.5 % and resembles the situation for D. It may be concluded that bonding of several functional groups or a single group but of higher mass to the furanose ring lowers the strength of the cation ring and fragmentation via ring rapture becomes more favourable. It is also of note that photofragmentation of D in the gas phase [13] produces similar groups of cations to that obtained in electron impact ionization, although with different relative abundances of the individual groups.

Fragmentation processes

The (

) 2
In both cases it requires scission of the C(5)-O(1) and C(2)-C(3) bonds within the ring (see figure 1a). The AE obtained for the C 3 H 6 + cation is equal to 11.4 ± 0.25eV (figure 3) and agrees well with the value of Ref. [24]. It coincides with the vertical ionization potential of the 11a/11aó rbital (11.37 eV [35]), while the AE of C 3 H 6 + of 10.65 ± 0.2 eV obtained in photoionization measurements [29] agrees well with the adiabatic ionization energy of the 11a/11a´orbital. This may suggest, in both electron impact and photoionization, a reaction channel where an electron is removed from the 11a/11a´(n O|| , CC ) orbital. This would weaken the respective C-O and C-C bonds and initiate dissociation of the C 4 H 8 O + ion.

The second most abundant cation (mass 41 amu) corresponds to C 3 H 5 + in both conformations of linear and cyclopropenyl ions [24,[START_REF] Duffield | [END_REF]. The neutral products are the methoxy, CH 3 O radical or the formaldehyde molecule and an H atom [24] giving the two following reactions

C 4 H 8 O + C 3 H 5 + + •CH 3 O and C 4 H 8 O + C 3 H 5 + + CH 2 O + H. ( 3
)
The first reaction requires rearrangement of the H atoms in the THF ion [24]. The present AE is lower than that from two previous electron impact measurements [23,24] and is just above the value obtained in the photoionization studies [29] (see Table 2). The efficiency curve in the threshold region was fitted with an exponential function with n=2.2 (figure 3) which is higher than for the case of Ar + , where n=1. Higher values of n correspond to a slower increase of the ionic cross section in the threshold region, which may result from excitation of the internal energy levels (vibrational and electronic) of the cation. The C 3 H 5 + ionic fragment is again formed by breaking of the C-C and C-O bonds in the furanose ring.

The most obvious reactions producing cations of the masses 43 and 44 amu are

C 4 H 8 O + C 2 H 3 O + + C 2 H 4 + H and C 4 H 8 O + C 2 H 4 O + + C 2 H 4 , (4)
respectively, both involving the release of a neutral ethylene molecule. The AE of the C 2 H 3 O + cation, 10.90 ± 0.25 eV is equal to a value obtained in the photoionization investigation [START_REF] Duffield | [END_REF] and is lower than the earlier electron impact results [23,24]. The value of n=2.2 obtained in the efficiency curve fitting again points at a possible excitation of the internal energy levels of the cation. The 39 and 40 amu peaks in the mass spectrum correspond to the C 3 H

The 29 and 31 amu peaks are assigned to the CHO + and CH 3 O + cations, respectively. Here, a similarity with the tetrahydrothiophene, C 4 H 8 S molecule was exploited. For this molecule, the deuterium labeling method [START_REF] Duffield | [END_REF] showed the origins of the corresponding CHS + and CH 3 S + cations. The 15 amu peak is due to the methyl CH 3 + cation. The above low-mass cations involve multiple bonds rearrangement and hydrogen atoms transfer along the ring in the processes of fragmentation. The present AEs of the ionic fragments in the 15-40 amu range are lower than the earlier electron impact values [23] and are closer to the corresponding photoionization results [START_REF] Duffield | [END_REF].

Partial and total ionic efficiency curves

The ionic efficiency curves of the THF fragments recorded in the mass spectrum of figure 2 were measured from 5 to 150 eV. These curves were normalized to each other according to abundances at 140 eV given in Table 1. Figure 4 displays examples of the curves obtained for cations of 72, 44, 42, 26 and 15 amu, respectively. Sum of the partial efficiency curves of the individual ionic fragments represents the relative total ionization cross section. Figure 5 compares the total ionization cross section with the results of binary-encounter-Bethe calculations [28]. This approach predicts the total ionization cross section as the sum of the ionization cross sections for removing one electron from each of the molecular orbitals that contribute to ionization [36]. The ionization cross section is given by a simple analytic expression, as a function of incident electron energy, which does not include details of ionization routes. For comparison both results are normalized to unity at their maxima. There is a general agreement between the experimental and calculated cross sections, although the experimental curve rises faster above threshold and displays a maximum at a lower electron energy of 58.5 eV.

Conclusions

This work reports measurements of the cation mass spectra, ionization and ionic fragmentation efficiency curves of the THF molecules in the electron energy range 5-150 eV using mass spectrometry technique. A number of well resolved mass peaks were detected in the mass range 10-72 amu and assigned to the corresponding ionic molecular fragments. The mass spectra are in good agreement with the NIST data. The present spectra resolved some inconsistencies between NIST data and the mass spectra published very recently [27]. The AEs of the identified cations were also determined. The present values of AEs do not confirm some of the early electron impact measurements. The cation efficiency curves measured over the 5-150 eV range were normalized to each other with a reference to the mass spectra to determine their contributions to the total ionization cross section. The sum of the cation efficiency curves, which gives the relative total ionization cross section is compared with the binary-encounter-Bethe calculations [28]. There is a general agreement between the experimental and calculated cross sections, although the experimental curve rises faster above threshold and displays a maximum at a lower electron energy. These differences may further indicate that the binary-encounter-Bethe method can not give account of ionic fragmentation in ionization.

The cation mass spectrum of the THF is compared with that of D. It is concluded that in the case of ionization and ionic fragmentation THF has some limits to be considered as an equivalent analogue of D. This conclusion corroborates that of the dissociative electron attachment studies which "demonstrated that THF cannot be used as a surrogate to model deoxyribose in DNA with respect to the attack of electrons at subexcitation energies (<3 eV)" [21]. The total ionization cross sections of tetrahydrofuran molecules obtained in the electron energy range 5-150eV in the present measurements and in the binary-encounter-Bethe calculations of Mo ejko and Sanche [28]. For comparison both curves are normalized to unity at their maxima.

Phys. Lett. 264 149 [Figure captions Figure 1 .

 149captions1 Figure captions

Figure 2 .

 2 Figure 2. The mass spectrum of cations of tetrahydrofuran molecules recorded at an electron energy of 140eV. The inset shows in detail the mass range from 48 to 60 amu.

Figure 3 .

 3 Figure 3. The ionic efficiency curves for cations of m = 71, 42, 41, 29 amu of tetrahydrofuran molecules measured in the near-threshold regions. The solid lines show curves of the indicated exponential functions fitted to the experimental data. The appearence energies are indicated by arrows.

Figure 4 .

 4 Figure 4. The ionic efficiency curves for cations of m = 72, 44, 42, 26, 15 amu of tetrahydrofuran molecules measured in the electron energy range 5-150eV.

Figure 5 .

 5 Figure 5. The total ionization cross sections of tetrahydrofuran molecules obtained in the electron energy range 5-150eV in the present measurements and in the binary-encounter-Bethe calculations of Mo ejko and Sanche[28]. For comparison both curves are normalized to unity at their maxima.

Fig. 1

 1 Fig.1

Fig. 2

 2 Fig. 2

  Fig.3

Fig. 5

 5 Fig.5

  and formation of a number of cationic fragments. The mass spectrum of THF cations measured at an incident electron energy of 140 eV is shown in figure2together with tentative assignments of the observed mass peaks. Spectra with nearly identical peak intensities were also obtained at 70 eV. The relative abundances of the cations seen in the main spectrum of figure 2 are listed in table 1 in percentage ratio to the most intense mass of 42 amu. The AEs, which were determined for the individual ionic fragments, are presented in table 2 as mean values derived from several determinations and are compared with results of other works[23,24,29].

  parent C 4 H 8 O + ion is formed by ionization from the highest occupied non-bonding oxygen atom lone-pair 9b/12a´(C 2 /C s THF conformations) orbital. Its abundance is about three times lower than that of the most intense ion peak at 42 amu. The AE obtained for C 4 H 8 O + ion is 9.55 ± 0.15 eV and this value is consistent with the previous electron impact[23,24] and photoionization[29, 34, 35] measurements as is shown in Table2. For comparison, the AE of the parent ion of D is 10.51 ± 0.11[12]. It is about 1 eV higher than that of THF. The C 4 H 7 O + cation (of mass 71 amu) involves electron detachment with simultaneous cleavage of one of the C-H bonds. The ionization takes place from the 9b/12a´orbital because the C 4 H 7 O

	Standard ab initio molecular orbital calculations [32] eliminated the possibility that the THF ion
	dissociates via a ring-opening reaction leading to the distonic •CH 2 CH 2 CH 2 OCH 2	+ isomer.
	The most abundant ion (mass 42 amu) is identified as the cyclopropane C 3 H 6 + ion radical
	from measurements in deuterium THF derivative [26] and estimations of heat of formation of
	C 3 H 6 + [24]. The neutral fragment with mass 30 amu is the formaldehyde, CH 2 O molecule,
	however formation of CO and H 2 cannot be excluded. This suggests the following reactions
	C 4 H 8 O + C 3 H 6	+ + CH 2 O and C 4 H 8 O + C 3 H 6	+ + CO + H 2 .

+ AE of 10.20 ± 0.25 eV (figure

3

) is lower than the adiabatic ionization energy (10.7 eV) of the next occupied 11a/11a´orbital

[34]

. The deuterium labeling method

[29]

established that 70% of the ions correspond to loss of an -H atom and the rest to loss of an -H atom by the THF molecule.

Table 1 .

 1 Abundances and assignments of cations of tetrahydrofuran molecules measured at an electron energy of 140 eV.

	m	Cation	Relative abundance
	(amu)	assignment
	72	C 4 H 8 O +	32%
	71	C 4 H 7 O +	30%
	44	C 2 H 4 O +	4%
	43	C 2 H 3 O +	19%
	42	C 3 H 6 +	100%
	41	C 3 H 5 +	40%
	40	C 3 H 4 +	8%
	39	C 3 H 3 +	10%
	31	CH 3 O +	3%
	29	CHO +	9%
	28	C 2 H 4 +	5%
	27	C 2 H 3 +	17%
	26	C 2 H 2 +	3%
	15	CH 3	+	4%

Table 2 .

 2 Appearance energies (in eV) of cations of tetrahydrofuran molecules obtained in the present work, compared with electron impact (EI) and photoionization (PI) measurements.

	m (amu)	Present work	Meyer et al PI	Gallegos and Kiser EI	Collin and Conde-Caprace EI
	72	9.55 ± 0.15	9.445 ± 0.010	9.45 ± 0.15	9.55
	71	10.20 ± 0.25	<10.0 ± 0.1	11.1 ± 0.2	10.44
	45		12.15 ± 0.10
	44		11.0 ± 0.1		12.27
	43	10.90 ± 0.25	10.9 ± 0.2	12.8 ± 0.2	11.87
	42	11.40 ± 0.25	10.65 ± 0.20	12.7 ± 0.2	11.54
	41	12.9 ± 0.2	12.7 ± 0.2	15.5 ± 0.3	13.72
	40	13.50 ± 0.25	12.9 ± 0.2	15.2 ± 0.3
	39	15.6 ± 0.2	14.9 ± 0.2	18.7 ± 0.6
	38		23.5 ± 0.2
	37		25 ± 1
	31	12.0 ± 0.2	11.85 ± 0.10
	29	11.65 ± 0.20	12.5 ± 0.2	15.8 ± 0.2
	28	13.95 ± 0.25	12.5 ± 0.2
	27	13.30 ± 0.25	13.3 ± 0.1	16.1 ± 0.3
	26	11.40 ± 0.25	14.9 ± 0.2	17.3 ± 0.3
	16		15.6 ± 0.1
	15	14.0 ± 0.5	16.0 ± 0.3
	14		19.5 ± 0.5

Acknowledgements

This work was carried out within COST Action CM0601 "Electron Controlled Chemical Lithography". It was supported by the Polish Ministry for Science and Higher Education under contract 553/N-COST/2009/0 .


Keywords: adolescent < Age, child < Age, blood transfusion < Hematology, acute < Pain, scoliosis < Orthopedics, survey

Blood conservation and pain control in scoliosis corrective surgery: an online survey of UK practice.

Background. Following discussions at national meetings the authors realised the diverse range of anaesthetic practice, in paediatric and adolescent scoliosis surgery, current in the U.K. This coupled with the paucity of research into the peri operative care of the scoliosis patient led to this national survey. We assessed this diversity using an online survey, with particular reference to pain control, thromboprophylaxis and blood conservation strategies. The aim of the survey was to provoke discussion and highlight areas of future research.

Methods.

The names of anaesthetists specialised in paediatric and adolescent scoliosis surgery in the U.K. were collated and contacted via email, asking them if they would fill in a short email survey. Non responders were contacted a total of three times over a 6 month period. Of the 24 centres practicing paediatric and adolescent scoliosis surgery we received completed questionnaires from anaesthetists working in 21. A response rate of 88%.

Results.

Blood Conservation 20 centres (95%) routinely cross-match blood. 17 centres (81%) routinely use cell salvage with a further 3 (14%) using it for specific cases. Only 4 centres (19%) advocated normovolaemic heamodilution, with no centres surveyed using pre donated blood. (figure 1) Peri-operatively, 17 centres (81%) routinely used anti-fibrinolytics, all of whom use Tranexamic Acid. Reported dosing regimens showed an even wider variation than previously reported (1) with loading doses varying between 2-100mg/kg and infusions varying from 3-10mg/kg/hr.

Thromboprophylaxis

Three centres (14%) used graduated compression devices (TEDS) alone, 3 centres (14%) TEDS and foot pumps, 6 centres (29%) foot pumps alone and 8 centres (38%) used no mechanical devices. Low molecular weight heparin (LMWH) was only used as part of a thromboprophylaxis regime in 6 centres (29%) (figure 2)

Those centres that advocated the use of LMWH showed wide variation in timing of therapy; some centers prescribing it the night before, others post operatively on the day of surgery, some 24 hours post surgery and one centre on day 2.

Figure2. Thromboprophylaxis strategies utilized by U.K. centres.

Pain Control

Regional: Epidural analgesia is routinely used in 8 centres (38%), of which 3 centres (14%) place 2 catheters and 2 centres (10%) use patient controlled epidural analgesia, all continue use

Institutional factors

The majority of centres have 2-3 scoliosis surgeons (range 1-5). Of whom most undertake posterior repairs more frequently than either anterior or anterior-posterior combined. There was

Preoperative investigation

Whilst 20 centres (95%) routinely cross-match blood, 14 centres (67%) would order pulmonary lung function tests, 2 centres (10%) routinely order arterial blood gases and 3 centres (14%) Echocardiograms with one centre (5%) ordering none of the above.

Perioperative anaesthetic techniques

17 centres (81%) utilised hypotensive anaesthesia with 5 of these (29%) defining this as an active process using a combination of agents including remifentanyl, labetolol, GTN, clonidine and vapour.

The maintenance agent of choice was divided almost equally between vapour, 9 centres (43%) and TIVA, 12 centres (57%) techniques.

With regards intra operative monitoring, all centres routinely place arterial lines and 16 centres (76%) CVP lines. 3 centres (14%) also use oesophageal Doppler and one utilises LiDCO.

For intra operative spinal cord monitoring SSEP was used in 19 centres (90%) although other monitors are also utilised. BIS or other such devises were used in only 2 centres.

Discusion.

The results from our survey show the expected variation in numbers and types of procedures undertaken across the centres in the U.K. As was expressed in the invitation to undertake the survey our intent was not to promote conformity but to stimulate debate.

Blood conservation. The area of greatest clinical variability seen in this survey was the dosing regimens used for Tranexamic Acid. With loading doses varying between 2-100mg/kg and infusions varying from 3-10mg/kg/hr. The Cochrane database review of Antifibrinolytic agents for reducing blood loss in scoliosis surgery in children in 2008 [START_REF] Tzortzopoulou | Antifibrinolytic agents for reducing blood loss in scoliosis surgery in children[END_REF] stated that antifibrinolytic drugs reduced blood loss and the amount of blood transfused. However the fact that only six studies met their inclusion criteria and that no comment on dosing regimens was made shows that there is scope for further research into this area.

Thromboprophylaxis. Another area of diversity demonstrated in our survey was that of thromboprophylaxis. Not only was there a broad range of devices used, some in isolation and others in combination, some centres reported not using any mechanical compression pre or post operatively. LMWH were used in only 6 centres and even then showed great variation in their time of administration. The evidence again is sparse and conflicting; a recent study [START_REF] Pateder | Pulmonary embolism after adult spinal deformity surgery[END_REF] embolism (PE) as 2.4%, with an increased incidence in those patients undergoing anterior spinal surgery. When comparing their results with an historical control group who had no thromboprophylaxis they concluded that pharmacological thromboprophylaxis probably doesn't have a role after posterior spinal surgery but could lower the incidence of PE after anterior spinal surgery. A recent pilot [START_REF] Kaabachi | Screening for deep venous thrombosis after idiopathic scoliosis surgery in children: a pilot study[END_REF] which screened children for deep vein thrombosis (DVT) after posterior spinal fusion for idiopathic scoliosis, looked at 40 patients and demonstrated two transient small clots. The authors stated that prophylaxis for venous thrombosis should not be recommended. However they added that larger series were required, they did not mention anterior spinal surgery.

Pain control. The frequency and severity of post op pain was not reviewed in this survey, however the survey showed that eight different types of post operative pain relief were utilised, some in isolation but many in combination which perhaps is evidence of the potential difficulties in treating post operative pain in this cohort of patients. A recent meta-analysis [START_REF] Taenzer | Efficacy of postoperative epidural analgesia in adolescent scoliosis surgery: a meta-analysis[END_REF] looked at the evidence to recommend the use of epidural analgesia with parenteral opioids vs intravenous opioids only. The authors stated that epidural analgesia is beneficial to patients in terms of improving pain control and reducing side effects, however there was not enough data available to state the impact of this technique on respiratory depression, length of stay or mortality. With the suggestion that these patients may be presenting with chronic pain (6) later in life the need for research into the best perioperative analgesic regime is even more pressing.

  Blood conservation and pain control in scoliosis correctivesurgery: an online survey of UK practice. S.Bird¹ and N. McGill 1 ¹Department of Anaesthesia Southampton University Hospital, Southampton SO16 6YD.

Figure 1 .

 1 Figure 1. Blood conservation strategies utilized by U.K. centres.

  Figure3. Post operative pain relief strategies utilized by U.K. centres.

  spread when looking at total cases undertaken per annum. With 2 centres (10%) undertaking <25 cases, 7 centres (33%) undertaking 25 -50 cases, 7 centres (33%) undertaking 50 -100 cases, 4 centres (19% ) undertaking 100 -200 cases and only one centre (5%) reporting over 200 cases a year. There is facility for High care beds (HDU and or ITU) in 90 % of the centres.

  86%) extubate their patients in theatre with 2 centres (10%) experiencing respiratory failure, as defined by PaO2<8.0 or PaCo2>6.0, in the first 24 hours in more than 5% of their patients.

  looked at adult patients undergoing spinal deformity surgery who all received post operative pharmacological thromboprophylaxis. Here the authors stated an overall incidence of pulmonary

.

Conclusion

  The results from our survey show wide variation in blood conservation, thromboprophylaxis and pain control techniques in scoliosis correction surgery in the U.K. National multi-centered research is required to define best practice.Key words:Survey, Anaesthetic practice, paediatric, scoliosis surgery, blood loss, pain control VV, Caltoum C, Petcharaporn M, Bastrom TP, Pawelek JB, Betz RR, Clements DH, Lenke LG, Lowe TG, Newton PO. Adolescent idiopathic scoliosis patients report increased pain at five years compared with two years after surgical treatment. Spine (Phila Pa 1976). 2008 May 1;33(10):1107-12.

Appendix A

Hospital taking part in survey, alphabetical order.

Addenbrookes