Phenyl N-Tert-Butyl Nitrone Forms Nitric Oxide as a Result of Its Fe(Iii)-Catalyzed Hydrolysis Or Hydroxyl Radical Adduct Formation

Phenyl N-tert-butyl nitrone (PBN) is commonly employed in spin-trapping studies. We report here evidence that PBN in aqueous solutions is decomposed by two pathways leading to the generation of nitric oxide ('NO). The first pathway is by hydrolysis of PBN, which is strongly catalyzed by ferric iron. The second pathway is via PBN-hydroxyl radical adduct formation. NO was trapped in the presence of cysteine and ferrous iron to form a [(cys)₂ Fe(NO)₂] ^-3 complex, which was measured by use of electron paramagnetic resonance (EPR) spectroscopy. A concomitant metabolite, benzaldehyde, was detected from both reaction mixtures. We propose that PBN is hydrolyzed by Fe³⁺ or attacked by hydroxyl radical, leading eventually to a common transient species, tert-butyl hydronitroxide [t-BuN(O')H], which is further oxidized to a 'NO source, t-BuNO. Our data imply that PBN may decompose to 'NO when used in biological models with oxidative stress conditions.     

Dinitrosyl Iron Complexes with Thiol-Containing Ligands Exist in Living Organisms Mainly in the Binuclear Form

The levels of the mononitrosyl iron complex with diethyldithiocarbamate that form in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite, or the vasodilating drug isosorbide dinitrate (Isoket®) have been assessed by electron paramagnetic resonance (EPR). The levels of the complex in mice that received binuclear dinitrosyl iron complexes with thiol-containing ligands or S-nitrosoglutathione do not change after the treatment of liver preparations with the strong reducing agent dithionite, in contrast to those formed after nitrite or isosorbide dinitrate administration, whose levels sharply increase after the same treatment. It is inferred that in the latter case an EPR-active mononitrosyl iron complex with diethyldithiocarbamate is produced with the absence or presence of dithionite in the reaction of NO formed from nitrite with Fe²⁺-diethyldithiocarbamate and Fe³⁺-diethyldithiocarbamate complexes, respectively. In the former case, the mononitrosyl iron complex with diethyldithiocarbamate is produced by transition of iron-mononitrosyl fragments from already present iron-dinitrosyl groups of binuclear dinitrosyl complexes, whose content is three to four times higher than the content of the mononuclear form of these complexes in the tissue. The results we obtained indicate that when dinitrosyl iron complexes with thiol-containing ligands, either introduced into the body or produced with the participation of endogenous NO, appear in animal tissues in vivo, these complexes are presented in these tissues mainly in their diamagnetic, EPR-silent binuclear form.

     

Gamma-irradiation potentiates L-arginine-dependent nitric oxide formation in mice.

Gamma-irradiation of mongrel mice at a sublethal dose (700 Roentgen) enhanced the formation of nitric oxide (NO) in the liver, intestine, lung, kidney, brain, spleen or heart of the animals. NO formation was determined by the increase in intensity of the EPR signal due to trapping of NO into mononitrosyl iron complexes (MNIC) with exogenous diethyldithiocarbamate (DETC) injected intraperitoneally. The EPR signal of these MNIC-DETC complexes was characterized by g-factor values at g perpendicular values at g perpendicular = 2.035 and g parallel = 2.02 and a triplet hyperfine structure at g perpendicular. The NO synthase inhibitor, NG-nitro-L-arginine, prevented MNIC-DETC complex formation both in liver and intestine, demonstrating the involvement of endogenous NO formed. Thus, gamma-irradiation may enhance endogenous NO biosynthesis in these tissues, presumably by facilitating the entry of Ca2+ ions into the membrane as well as the cytosol of NO-producing cells through irradiation-induced membrane lesions.     

Characteristics and use as spin trapping agent of a beta-phosphorylated nitroso compound, DEPNP

2-(Diethylphosphonate)-nitrosopropane (DEPNP), prepared by oxidation of the corresponding aminophosphonate, was found to essentially exist as monomer in both water and organic solvents. The mechanisms of its degradation under 80°C heating or visible light exposure were studied by EPR spectroscopy: its decomposition gave rise to paramagnetic by-products, which have been identified as DEPNP / ·C(CH₃)₂[P(O)(OC₂H₅)₂] and DEPNP / ·P(O)(OC₂H₅)₂ spin adducts. Despite this drawback, DEPNP was successfully used as spin trapping agents to scavenge various carbon — and phosphorus-centred free radicals both in aqueous and organic media, giving rise to intense EPR spectra characteristic of the species trapped.     

Intracellular free iron in liver tissue and liver homogenate: studies with electron paramagnetic resonance on the formation of paramagnetic complexes with desferal and nitric oxide.

Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.     

Inhibitory effect of nitric oxide on the induction of cytochrome P450 3A4 mRNA by 1,25-dihydroxyvitamin D3 in Caco-2 cells

Cytochrome P450 (CYP)-dependent drug metabolism decreases in vivo and in cultured hepatocytes under various immunostimulatory conditions. Nitric oxide (NO) released during inflammation is presumed to be involved in this phenomenon. CYP3A4, which is abundant in the liver and small intestine and participates in the metabolism of various drugs, is known to be induced by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in the colon carcinoma cell line Caco-2. In this study we examined whether NO affected CYP3A4 gene expression induced by 1,25(OH)₂D₃ in Caco-2 cells. Induction of CYP3A4 mRNA by 1,25(OH)₂D₃ was suppressed in a dose-dependent manner by treatment with the NO donors NOR-4 (15-500 μM) or S-nitroso-N-acetyl-penicillamine (30 μM-1 mM), which spontaneously release NO. These results indicated that NO has an inhibitory effect on the induction of CYP3A4 mRNA by 1,25(OH)₂D₃ in Caco-2 cells. Treatment with the guanylate cyclase inhibitor ODQ failed to prevent the inhibition of induction of CYP3A4 mRNA by 1,25(OH)₂D₃. 8-Bromo cGMP had no effect on 1,25-(OH)₂D₃-induced CYP3A4 gene expression. Therefore, the suppression of CYP3A4 mRNA by NO might be mediated through a guanylate cyclase-independent pathway.     

Synthesis and structures of the cuboidal iron-sulfur-nitrosyl-phosphine clusters [Fe4S3(NO)4(PR3)3] (R = Et, Pr, C6H11)

A series of cuboidal iron-sulfur clusters [Fe₄S₃(NO)₄(PR₃)₃]^0,1+ (R = Et, Prⁱ, Cy) were synthesized by two routes: reductive desulfurization of [Fe₄S₄(NO)₄] by tertiary phosphines, and substitution of triphenylphosphine in [Fe₄4S₃(NO)₄(PPh₃)₃] by a more basic phosphine. The structures of 3[Fe₄S₃(NO)₄(PEt₃)₃] · 0.5Et₂O, [Fe₄S₃(NO)₄(PEt₃)₃] [Fe₄S₃(NO)₇] and partially substituted [Fe₄S₃(NO)₄(PPh₃)_2⁻ (PPrⁱ₃)] have been determined by X-ray diffraction in order to define the cuboidal Fe₄S₃ core, previously known only in Roussin's black anion and its reduced form, [Fe₄S₃(NO)₇7]^1−,2−, and as a part of the iron-molybdenum cofactor of nitrogenase.     

Silicon and iron levels in tissues of animals treated with a “ferrimagnetic ceramic” with oncotherapeutic potential (anti-tumor) value

The stability in a biological environment of an injectable cement with oncotherapeutic potential — consisting of a glass powder of SiO₂ (35.6%), CaO (42.4%), P₂O₅ (17%), Na₂O (5%) and 30% of its weight of Fe₃O₄ dissolved in (NH₄)₂HPO₄ plus NH₄H₂PO₄ — was evaluated referring to the release of silicon and iron. The experimental model was the rat, and organs (liver, kidney, spleen, lung, heart, and brain) of the implanted and control animals were collected for quantification of these elements by electrothermal atomization atomic absorption spectrometry methods. In most of the analysed organs no significant difference in the contents of silicon and iron between the implanted and the control animals was found.     

Molecular structure and reactions of azobenzene complexes with iron-lithium compounds

Reactions of azobenzene have been studied with heteronuclear iron-lithium compounds formed in the reaction of FeCl₃ with LiPh, one of the dinitrogen reducing systems of the Vol'pin type: Ph₄FeLi₄(OEt₂)₄ (1) and (H₂)FePh₄Li₄(OEt₂)₄ (2). The structures of the azobenzene complexes formed, (N₂Ph₂)₃FeLi₃(OEt₂)₃ (3) and (N₂Ph₂)₃FeLi₂(THF)₂ (4), as well as an ether-containing analog of the latter, (N₂Ph₂)₃FeLi₂(OEt₂)₂ (5), were determined by X-ray analysis of single crystals. Coordination of azobenzene at FeLi₃ and FeLi₂ clusters was shown to result in a sigificant elongation of the NN bond; partial cleavage of this bond on protolysis of the complexes resulted in the formation of hydrazobenzene and aniline. Magnetic susceptibility measurements and theoretic analysis of a similar model complex leads to the conclusion that the iron oxidation state in 3 may be considered between iron (I) and iron(III) (close to iron(I)), whereas in 4 and 5 it is close to iron(II).     

Magnetic properties of a cobalt(II) dimer of pseudo tetrahedral geometry [Co2{(CO)9Co3C---COO}5, C14H19N2H]

The crystalline ion pair [Co₂{OOC---CCo₃(CO)₉}₅, C₁₀H₆(N(CH₃)₂)₂H] (1) presents unusual magnetic properties. The X-band EPR spectrum of 1 at room temperature presents two unresolved bands at g=1.98 and 4.55. At a low temperature (20 K), the cluster of clusters 1 presents a complicated spectrum with an intense signal at 1700 G. The magnetic susceptibility of 1 was fit to a two spin S₁=S₂=3/2 Heisenberg model, with J=11.2 cm⁻¹ and a g value of 2.3. There is no field dependence of the magnetization, which suggests intramolecular coupling between the two tetrahedral centers of the cluster. Molecular orbital modeling indicates a sigma path of exchange between two topologically non-equivalent cobalt(II) centers.     

Synthesis and characterization of polypyridineruthenium(II) complexes containing a linear ambidentate ligand, NCO or NCS, and reaction of isocyanato complexes under acidic conditions

Isocyanato and isothiocyanatopolypyridineruthenium complexes, [Ru(NCX)Y(bpy)(py)₂]ⁿ⁺ (bpy=2,2′-bipyridine, PY=pyridine; X=O, Y=NO₂ for n=0, and Y=py for n=1; X=S, Y=NO₂ for n=0, Y=NO for n=2, and Y=py for n=1), were synthesized by the reaction of polypyridineruthenium complexes with potassium cyanate or sodium thiocyanate salt. Isocyanatoruthenium(II) complexes, [Ru(NCO)(NO₂)(bpy)(py)₂] and [Ru(NCO)(bpy)(py)₃]⁺, react under acidic conditions to form the corresponding ammineruthenium complexes, [Ru(NO)(NH₃)(bpy)(py)₂]³⁺. The molecular structures of [Ru(NCO)(bpy)(py)₃]ClO₄, [Ru(NCS)(NO)(bpy)(py)₂](PF₆)₂ and [Ru(NO)(NH₃)(bpy)(py)₂](PF₆)₃ were determined by X-ray crystallography.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

Structural determinations, magnetic and EPR studies of complexes involving the Cr(OH)2Cr unit

Five heterometallic compounds with formulae [Ba(H₂O)₄Cr₂(μ-OH)₂(nta)₂] · 3H₂O (I), [M(bpy)₂(H₂O)₂] [Cr₂(OH)₂(nta)₂] · 7H₂O, where M²⁺ = Zn, (II); Ni, (III); Co, (IV) and [Mn(H₂O)₃(bpy)Cr₂(OH)₂(nta)₂] · (bpy) · 5H₂O (V); bpy = 2,2′-bipyridine, (nta = nitrilotriacetate ion) have been prepared by reaction of I with the corresponding M^II-sulfates in the presence of 2,2′-bipyridine. Substances I–V have been characterized by magnetic susceptibility measurements, EPR and X-ray determinations. I represents a 2D coordination polymer formed by coordination of centrosymmetrical dimeric chromium(III) units and Barium cations. The 10-coordinate Ba polyhedron is completed by four water molecules. Compounds II–IV are isostructural and consist of non-centrosymmetric dimeric anions [Cr₂(μ-OH)₂(nta)₂]²⁻, complex cations [M^II(bpy)₂(H₂O)₂]²⁺ and solvate water molecules. The octahedral coordination of chromium atoms implies four donor atoms of the nta³⁻ ligands and two bridging OH groups. Multiple hydrogen bonds of coordinated and solvate water molecules link anions and cations in a 3D network. A similar [Cr₂(μ-OH)₂(nta)₂]²⁻ unit is found in V. The bridging function is performed by a carboxylate oxygen atom of the nta ligand that leads to the formation of a trinuclear complex [Mn(bpy)(H₂O)₂Cr₂(μ-OH)₂(nta)₂]. Experimental and calculated frequency and temperature dependences of EPR spectra of these compounds are presented. The fine structure appearing on the EPR spectra of compound V is analyzed in detail at different temperatures. It is established that the main part of the EPR signals is due to the transitions in the spin states of a spin multiplet with S = 2. Analyses of experimental and calculated spectra confirm the absence of interaction between metal ions (M^II) and Cr-dimers in complexes III and IV and the presence of weak Mn–Cr interactions in V. The temperature dependence of magnetic susceptibilities for I–V was fitted on the basis of the expression derived from isotropic Hamiltonian including a bi-quadratic exchange term.     

Electrophilic abstractions on Os(H)2X(NO)L2: selective access to new unsaturated cationic Os(II) hydrides

Nitrosylation of Os(H)₃ClL₂ (L = P¹Pr₃) affords the known Os(H)₂Cl(NO)L₂ (2). Soft electrophiles (Ag⁻, Na⁻) react with complex 2 by chloride abstraction to ultimately yield truly 16-electron dihydride Os(H)₂(NO)(P¹Pr₃)₂⁻ (4a), characterized by variable-temperature NMR. Complex 4a reversibly binds H₂, forming Os(H)₂(H₂)(NO)(P¹Pr₃)⁻ with an unusually high barrier for intramolecular hydride exchange. Under kinetic conditions, protonation of 2 with strong acids follows the selectivity for chloride abstraction. Thermodynamically, protonation at the hydride is preferred, quantitatively producing cationic OsHCl(NO)L₂⁺, isolated and characterized by X-ray diffraction as the BAr₄^F− salt (7) (Ar^F=3,5−(CF₃)₂C₆H₃). Structures of isoelectronic OsHCl(NO)(PH₃)₂ and OsHCl(CO)(PH₃)₂ were optimized with ab initio DFT (Becke3LYP) methods and compared to show the greater unsaturation of the metal in the cationic species. Both complexes, 4a and 7, are highly electrophilic and reversibly coordinate dichloromethane in solution. The observed reactivity patterns of the synthesized unsaturated hydrides are rationalized in terms of the determining influence of the ‘push-pull’ π-stabilization of the metal center.     

Dinitrosyl iron complexes--structure and biological functions

Formation of dinitrosyl iron complexes (DNICs), which can be described by general formula Fe(NO)2(L)2, where L is carbonyl-, nitrosyl- or imino- complexing ligand, was observed in many kinds of living organisms, in a wide spectrum of physiological conditions associated with inflammation, ischemia/reperfusion and cancer. Accumulation of DNICs coincides with intensified production of nitric oxide in macrophages, neurons, endothelial cells, Langerhans' cells and hepatocytes. Low-molecular thiol-containing DNICs (DNIC-(RS)2) show vasodilatory action and they are proposed to play a role of nitric oxide transducers and stabilizers. DNICs have been shown to modulate redox potential of the cell via inhibition of glutathione-dependent enzymes, such as glutathione reductase, S-transferase and peroxidase. Although there is a convincing experimental evidence for their NO and NO+ donating function, the nature of DNICs formed in biological systems, their stability and biological role is still a matter of discussion.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and 15N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

Charge transfer photochemistry of quadruply bonded ditungsten halophosphine complexes

The quadruple metal-metal bonded complexes, W₂Cl₄(PR₃)₄ (PR₃ = PMe₃, PMe₂Ph, PBu₃), photoreact in dichloromethane with near-UV excitation (λ>375 nm) to yield a mixed valence W₂(II,III) photoproduct. Electronic absorption and EPR spectra of photolyzed solutions are identical to those obtained from the thermal oxidation of W₂Cl₄(PR₃)₄ by PhICI₂, which is known to yield W₂Cl₅(PR₃)₃. Subsequent reaction of the photolyzed solution yields the oxidized, confacial biotahedral W₂(III,III) halophosphine. Analysis of the organic photoproduct reveals that the halocarbon solvent is reduced by one electron to yield the chloromethyl radical. When the radical is produced in low yields, hydrogen abstraction from solvent appears to be sufficiently efficient to compete with dimerization and only chloromethane is observed; however, at higher concentrations, the chloromethyl radicals couple to produce dichloroethane. Photoreaction is observed only with near-UV excitation of the LMCT absorption manifold of W₂Cl₄(PR₃)₄. At lower energy wavelengths, transient absorption spectroscopy shows the production of the ¹δδ^* excited state, which decays to ground state over times commensurate with the decay of ¹δδ^* luminescence. In hydrocarbon solutions, no transient intermediate or photochemistry is observed, indicating that the LMCT excited state, although capable of reducing a C---X bond, cannot activate the stronger C---H bonds of hydrocarbons. The photochemistry and transient absorption spectroscopy results of the W₂Cl₄(PR₃)₄ complexes are compared to our previous studies of the homologs.     

A reversible NO complex of Fe(TIM): an S = 1/2FeNO nitrosyl

[Fe(TIM)(CH₃CN)₂](PF₆)₂ (1) (TIM = 2,3,9,10-tetramethyl-1,4,8,11-tetraazacyclodeca-1,3,8,10-tetraene) forms a complex with NO reversibly in CH₃CN (53±1% converted to the NO complex) or 60% CH₃OH/40% CH₃CN (81±1% conversion). Quantitative NO complexation occurs in H₂O or CH₃OH solvents. The EPR spectrum of [Fe(TIM)(solvent)NO]²⁺ in frozen 60/40 CH₃OH/CH₃CN at 77 K shows a three line feature at g=2.01, 1.99 and 1.97 of an S=1/2FeNO⁷ ground state. The middle line exhibits a three-line N-shf coupling of 24 G indicating a six-coordinate complex with either CH₃OH or CH₃CN as a ligand trans to NO. In H₂O [Fe(TIM)(H₂O)₂]²⁺ undergoes a slow decomposition, liberating 2,3-butanedione, as detected by ¹H NMR in D₂O, unless a π-acceptor axial ligand, L=CO, CH₃CN or NO is present. An equilibrium of 1 in water containing CH₃CN forms [Fe(TIM)(CH₃CN)(H₂O)]²⁺ which has a formation constant K_CH3CN=320 M⁻¹. In water K_NOK_CH3CN since NO completely displaces CH₃CN. [Fe(TIM)(CH₃CN)₂]²⁺ binds either CO or NO in CH₃CN with K_NO/K_CO=0.46, sigificantly lower than the ratio for [Fe^II(hemes)] of 1100 in various media. A steric influence due to bumping of β-CH₂ protons of the TIM macrocycle with a bent S=1/2 nitrosyl as opposed to much lessened steric factors for the linear Fe---CO unit is proposed to explain the lower K_NO/K_CO ratio for the [Fe(TIM)(CH₃CN)]²⁺ adducts of NO or CO. Estimates for formation constants with [Fe(TIM)]²⁺ in CH₃CN of K_NO=80.1 M⁻¹ and K_CO=173 M⁻ are much lower than to hemoglobin (where K_NO=2.5×10¹⁰ M⁻¹ and K_CO=2.3×10⁷) due to a reversal of steric factors and stronger π-backdonation from [Fe^II(heme)] than from [Fe^II(TIM)(CH₃CN)]²⁺.     

Use of genetically modified mice to examine the skeletal anabolic activity of vitamin D

We employed genetically modified mice to examine the role of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on skeletal and calcium homeostasis. In mice expressing the null mutation for 25-hydroxyvitamin D 1 hydroxylase (1OHase^−/−), or the vitamin D receptor (VDR^−/−), 1,25(OH)₂D₃ and calcium were both required for optimal epiphyseal growth plate development, serum calcium and phosphorus alone were sufficient to mineralize skeletal tissue independent of 1,25(OH)₂D₃ and the VDR, and endogenous 1,25(OH)₂D₃ and the VDR were essential for baseline bone formation. In 2-week-old 1OHase^−/− mice and in 2-week-old mice homozygous for the PTH null mutation(PTH^−/−), PTH and 1,25(OH)₂D₃ were each found to exert independent and complementary effects on skeletal anabolism, with PTH predominantly affecting appositional trabecular bone growth and 1,25(OH)₂D₃ influencing both endochondral bone formation and appositional bone growth. Endogenous 1,25(OH)₂D₃ maintained serum calcium homeostasis predominantly by modifying intestinal and renal calcium transporters but not by producing net bone resorption. Administration of exogenous 1,25(OH)₂D₃ to double mutant PTH^−/−1OHase^−/− mice produced skeletal effects consistent with the actions of endogenous 1,25(OH)₂D₃. These studies reveal an important skeletal anabolic role for both endogenous and exogenous 1,25(OH)₂D₃ and point to a potential role for 1,25(OH)₂D₃ analogs in the treatment of disorders of bone loss.