Phosphatidylserine (PS) is a phospholipid essential for cell membrane composition and with high concentrations in the brain. PS is widely used as a dietary supplement due to its neuroprotective effects and the benefits shown to patients with Central Nervous System (CNS) disorders. This work aimed to identify critical aspects in the development of a quantitative NMR (qNMR) method using quantitative 31P-NMR (qPNMR) with triphenyl phosphate (TPP) as an internal calibrant (IC) for the analysis of PS samples. The findings indicate several pivotal aspects in the method development: i) sample preparation is a critical step for successful qNMR analysis; the addition of EDTA 0.2M (pH 7.2-7.5, adjusted with Cs2CO3) enhances the lineshape and, thus, the overall resolution of the spectra; ii) the molecular weight of the analyte significantly impacts the analytical outcomes; and iii) post-acquisition processing conditions, including window functions and integration criteria, have slight but noticeable effects on the quantitative outcomes, and should therefore be considered an integral part of any convention method for PS quantitation. To overcome this latter aspect, peak fitting was found to be an effective approach for reducing operator bias, especially in integration, and is viable due to the essential near-singlet nature of the 31P-NMR peak patterns for each individual chemical species.

PURPOSE:To demonstrate the feasibility of using chemical shift magnetic resonance (MR) imaging fat-water separation methods for quantitative estimation of transcatheter lipiodol delivery to liver tissues. MATERIALS AND METHODS:Studies were performed in accordance with institutional Animal Care and Use Committee guidelines. Proton nuclear MR spectroscopy was first performed to identify lipiodol spectral peaks and relative amplitudes. Next, phantoms were constructed with increasing lipiodol-water volume fractions. A multiecho chemical shift-based fat-water separation method was used to quantify lipiodol concentration within each phantom. Six rats served as controls; 18 rats underwent catheterization with digital subtraction angiography guidance for intraportal infusion of a 15%, 30%, or 50% by volume lipiodol-saline mixture. MR imaging measurements were used to quantify lipiodol delivery to each rat liver. Lipiodol concentration maps were reconstructed by using both single-peak and multipeak chemical shift models. Intraclass and Spearman correlation coefficients were calculated for statistical comparison of MR imaging-based lipiodol concentration and volume measurements to reference standards (known lipiodol phantom compositions and the infused lipiodol dose during rat studies). RESULTS:Both single-peak and multipeak measurements were well correlated to phantom lipiodol concentrations (r(2) > 0.99). Lipiodol volume measurements were progressively and significantly higher when comparing between animals receiving different doses (P < .05 for each comparison). MR imaging-based lipiodol volume measurements strongly correlated with infused dose (intraclass correlation coefficients > 0.93, P < .001) with both single- and multipeak approaches. CONCLUSION:Chemical shift MR imaging fat-water separation methods can be used for quantitative measurements of lipiodol delivery to liver tissues.

Complete analysis of the 1 H NMR spectrum of huperzine A, 1‐amino‐13‐ethylidene‐11‐methyl‐6‐aza‐tricyclo[7.3.1.0 2, 7 ]trideca‐2(7),3,10‐trien‐5‐one, a Lycopodium alkaloid and anti‐Alzheimer drug lead containing an ABCD(E)(MN)(OP)X 3 Y 3 –type system of 15 nonexchangeable proton spins, is reported for the first time, and earlier assignments are corrected. The complete 1 H parameter set of 11 chemical shifts clarifies the diastereotopism of both methylene groups, and provides a total of 38 observed H,H‐couplings including 31 long‐range ( 4–6 J) connectivities. The NMR data is consistent with the comparatively rigid alicyclic backbone predicted by molecular mechanics calculations, and forms the basis for 1 H NMR fingerprint analysis for the purpose of dereplication, purity analysis, and elucidation of structural analogs. Copyright © 2007 John Wiley & Sons, Ltd.

Hormone level differences are generally accepted as the primary cause for sexual dimorphism in animal and human development. Levels of low molecular weight metabolites also differ between men and women in circulating amino acids, lipids and carbohydrates and within brain tissue. While investigating the metabolism of blue crab tissues using Phosphorus-31 Nuclear Magnetic Resonance, we discovered that only the male blue crab (Callinectes sapidus) contained a phosphorus compound with a chemical shift well separated from the expected phosphate compounds. Spectra obtained from male gills were readily differentiated from female gill spectra. Analysis from six years of data from male and female crabs documented that the sex-specificity of this metabolite was normal for this species. Microscopic analysis of male and female gills found no differences in their gill anatomy or the presence of parasites or bacteria that might produce this phosphorus compound. Analysis of a rare gynandromorph blue crab (laterally, half male and half female) proved that this sex-specificity was an intrinsic biochemical process and was not caused by any variations in the diet or habitat of male versus female crabs. The existence of a sex-specific metabolite is a previously unrecognized, but potentially significant biochemical phenomenon. An entire enzyme system has been synthesized and activated only in one sex. Unless blue crabs are a unique species, sex-specific metabolites are likely to be present in other animals. Would the presence or absence of a sex-specific metabolite affect an animal's development, anatomy and biochemistry?

This reports the in vitro portion of a study designed to establish guidelines for the preparation, storage, and use of tribromoethanol (TBE). We evaluated: 1) the purity of TBE powder from three suppliers; 2) nine methods of preparation of a 25-mg/ml (working) solution for formation of particulates and breakdown products; 3) formation of particulates and breakdown products and pH change in 1-g/ml (stock) solutions and working solutions stored under four conditions (25 degrees C and 5 degrees C in light and in dark); and 4) stock and working solutions of TBE that caused lethal effects in mice. These objectives were met by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectroscopy, particle-size and turbidity analyses, and pH strips. TBE powder from three suppliers varied in purity. No significant differences in breakdown product formation, particle size, or turbidity were noted between the nine preparation methods evaluated. Stock solutions and the working solution stored at 5 degrees C in the dark maintained a pH of 6.5 to 7.0, whereas the pH dropped for all other working solutions. A low level of dibromoacetaldehyde (DBA), a potential breakdown product reported to cause toxic effects, was detectable in all newly prepared solutions. Regardless of the storage condition or pH, DBA concentration did not increase measurably in any of the solutions after 8 weeks. The stock and working solutions that demonstrated lethal effects in mice had a pH of 6.5 and did not differ notably from newly prepared, non-lethal solutions, when evaluated for DBA. A decrease in pH could not be correlated to an increase in DBA or potential lethality, as suggested in the literature. The toxicity associated with the lethal TBE in this study appears to be a result of a chemical reaction or breakdown product that has not yet been reported.

Alanine and lactate, as major gluconeogenic substrates, must be converted into oxaloacetate by way of pyruvate carboxylase before their entry into gluconeogenesis. Although it is well known that hepatic gluconeogenesis from these substrates is increased in tumor hosts, the involvement of pyruvate carboxylase has not been demonstrated. In the present study, we examined pyruvate carboxylase activity in the perfused livers of tumor rats using 13C NMR spectroscopy with [3-13C]-alanine as the gluconeogenic substrate. A substantial increase in hepatic [3-13C]-aspartate production was found in the tumor rats. Since aspartate accumulation directly reflects fluxes of alanine through pyruvate carboxylase, the observed increase in hepatic production of [3-13C]-aspartate in tumor rats indicates that pyruvate carboxylase activity is significantly enhanced.

The line widths of35Cl− nuclear magnetic resonances were used to measure chloride binding by Band 3. Since this procedure related directly to binding, the data obtained may be interpreted more unequivocally than affinities derived from kinetic data which could be related to either translocation or binding. Chloride binding to the active sites in Band 3 was assessed from that portion of the total line width which was sensitive to 4,4′-dinitrostilbene-2,2′-disulfonic acid. These sites appeared to be completely inhibited by treatment of erythrocyte membranes with diethylpyrocarbonate. This result is consistent with our previous observation that this reagent inhibits anion transport in resealed erythrocyte ghosts (Izuhara, Okubo & Hamasaki, 1989,Biochemistry28:4725–4728). Hydroxylamine could not reverse the diethylpyrocarbonate inhibition of chloride binding to Band 3. The pH-dependence of diethylpyrocarbonate reactivity suggests that the modified residues may be those of histidine.

A surface coil holder is described for the General Electric Signa spectrometer. This allows for accurate positioning of the 31P coil relative to that of the 1H coil used for tissue slice localization.

Intracellular pH is thought to play an important role in several aspects of cell function including urinary acidification. The turtle urinary bladder is capable of urinary acidification in vitro and it is considered an analogue of the mammalian distal nephron. In the present study we measured intracellular pH in the epithelial layer of the turtle urinary bladder utilizing phosphorus nuclear magnetic resonance. In 14 experiments, the intracellular pH of the stripped turtle bladder epithelium bubbled with compressed air was 6.91 +/- 0.02; in turtle red blood cells the mean intracellular pH was 6.97 +/- 0.05 (n = 20), a value not significantly different from the pH value obtained in the turtle bladder. Gassing the turtle bladder with 95% air and 5% CO2 while maintaining the extracellular pH constant resulted in a rapid decrease in intracellular pH. Lowering of extracellular pH with HCl also resulted in a decrease in intracellular pH. The results demonstrate that phosphorus nuclear magnetic resonance allows rapid, noninvasive measurement and frequent monitoring of intracellular pH in the turtle bladder.

In order to find a suitable marker of intraerythrocytic pH in a phosphorus-31 nuclear magnetic resonance (31P NMR) system, analogues of inorganic phosphate were studied. Fluorophosphate (pKa = 4.7) and phosphite (pKa = 6.4) chemical shifts and coupling constants were found to be pH sensitive but at pH ranges too low to be useful for the study of fresh red cells. Methylphosphonate (pKa = 7.6) showed ideal characteristics for a pH probe: its chemical shift was far downfield for red cell phosphates and showed a large pH dependence near its pKa (delta pH/delta delta = 0.46 pH/ppm). Methylphosphonate readily entered red cells [influx 7.8 microM (mL of RBC)-1 h-1] and did not appear to alter glucose consumption or hemoglobin-oxygen affinity in intact cells. NMR spectra were obtained on eight samples of fresh red cells incubated for 30-60 min with methylphosphonate. Chemical shift differences between the extracellular and intracellular methylphosphonate signals were found to be predictive of the transmembrane pH gradient. The extracellular pH was 7.336 +/- 0.031 (range 7.31-7.41), while the intracellular pH was 7.202 +/- 0.034 (range 7.14-7.23), and the transmembrane pH gradient measured 0.129 +/- 0.008 (range 0.11-0.14). Methylphosphonate is a useful probe of pH in the 31P NMR spectroscopic study of red cells.

Skeletal muscle contains appreciable lysophospholipase activity which is differentiated by muscle type with red muscle subcellular fractions having greater activity than the corresponding white ones.

The effect of intracellular calcium on intracellular pH in the turtle urinary bladder was examined with phosphorus nuclear magnetic resonance. The turtle urinary bladder is capable of acidification in vitro and urinary acidification by this membrane is inhibited by an increase in intracellular calcium. Since calcium is capable of altering intracellular pH, it remains unclear whether the inhibition of urinary acidification is the result of an increase in intracellular pH. In the present study, intracellular calcium was increased by the cholinergic agent, carbachol, the ionophore A23187 and replacement of extracellular Na by sucrose. All agents decreased intracellular pH in the turtle bladder, thus suggesting that inhibition of urinary acidification by these agents is not due to an increase in intracellular pH.

Detailed analysis of appropriate 31P nuclear-magnetic-resonance spectra shows that under the usual laboratory conditions, carbodiimide-induced condensation of orthophosphoric acid in a number of solvents leads to condensation only slightly beyond the metaphosphate composition in the presence of strong tertiary amines; whereas in the absence of amine, the condensation proceeds into the ultraphosphate region about halfway between the metaphosphate and phosphoric anhydride compositions. With amine, the principal product consists of the cyclic trimetaphosphate anion, with one of the nonbridging oxygen atoms substituted by the urea resulting from hydration of the carboiimide, i.e., [N[CH(CH3)2] [C(O)NHCH(CH3)2]] for the condensation with diisopropylcarbodiimide. Without amine, the major product is the 1,5-μ-oxotetrametaphosphate anion . The well-known carbodiimide-mediated phosphorylation of alcohols with orthophosphoric acid is shown to be directly attributable to the high reactivity of the phosphate branch groups of the carbodiimide-generated ultraphosphates.

The phosphorus-containing side chains of two methylene-bridged analogs of adenosine triphosphate have been cyclized to produce the corresponding analogs of monoadenosine-5'-trimetaphosphate. (The structures are given in the journal.) The molecules, which were generated in anhydrous media through a carbodimide-mediated condensation, were characterized by 31P nuclear-magnetic resonance, and the white-noise 1H decoupled spectra were simulated. These molecules are quite reactive and readily converted to their corresponding linear forms upon hydrolysis. The second structure contains an asymmetric phosphorus atom, and both of the possible cyclic molecules have been observed and the diasteroisomeric mixture has been isolated.