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A polemic is given regarding several of the calculated curve-fit parameters that Zhou and coworkers reported in their published paper. The calculated curve-fit parameters for the Combined Nearly Ideal Binary Solvent/Redlich-Kister, Jouyban-Acree-van't Hoff, Sun and modified Apelblat models were found to give mole fraction solubilities that exceeded unity. Our analysis also found that the mean relative absolute percent deviations provided by the authors were significantly underestimated., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Human breast milk is the primary source of choline and choline-containing compounds for infants at early stages of life. Choline data across lactation in Chinese human milk were limited., Objective: This study aimed to quantify the five choline compounds in Chinese human breast milk and explore associated factors., Methods: A total of 540 lactating mothers from the MUAI (Maternal Nutrition and Infant Investigation) study were included. The content of water-soluble choline (free choline, phosphocholine, glycerophosphocholine) and lipid-soluble choline (phosphatidylcholine, sphingomyelin) in 892 human milk samples collected from 0 to 400 days postpartum were examined, and associated factors were explored., Results: Choline concentrations in human milk varied from postpartum day 0-400 (92.06 ± 65.22 to 171.01 ± 47.84 mg/L). Water-soluble choline was the major component (88.6%-93.8%) in human milk and ranged from 793.03 (659.22) to 1544.43 (443.32) μmol/L. Its trajectory followed that of total choline, increasing from colostrum to transitional milk and then declining in mature milk. In contrast, lipid-soluble choline accounted for 6.2%-11.4% over lactation and had an opposite trajectory. Choline composition varied by delivery mode and parity history., Conclusion: The concentrations of individual choline and choline-containing compounds during lactation in Chinese human breast milk were described for the first time. Our results address gaps in extant Chinese human milk choline data and support tailored dietary reference intakes for Chinese lactating women and infants. Our data describes the level and profile of choline from 0 to 400 days postpartum in Chinese human breast milk. This is the most updated data on choline and also the first report of water-soluble choline as the predominant type in Chinese human milk. Our results compensate for the deficiencies in data on choline in Chinese human milk., Clinical Trial Registration: Clinical Trial Registry number: ChiCTR1800015387. Web link to study on registry: https://www.chictr.org.cn/index.aspx., (Copyright © 2023 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

High-resolution magic-angle-spinning 1 H NMR spectroscopy (HR-MAS NMR) is a well-established technique for assessing the biochemical composition of intact tissue samples. In this study, we utilized a method based on HR-MAS NMR spectroscopy with slice localization (SLS) to achieve spatial resolution of metabolites. The obtained 7 slice spectra from each of the model samples (i.e., chicken thigh muscle with skin and murine renal biopsy including medulla (M) and cortex (C)) showed distinct metabolite compositions. Furthermore, we analyzed previously acquired 1 H HR-MAS NMR spectra of separated cortex and medulla samples using multivariate statistical methods. Concentrations of glycerophosphocholine (GPC) were found to be significantly higher in the renal medulla compared to the cortex. Using GPC as a biomarker, we identified the tissue slices that were predominantly the cortex or medulla. This study demonstrates that HR-MAS SLS combined with multivariate statistics has the potential for identifying tissue heterogeneity and detailed biochemical characterization of complex tissue samples.

Purpose: Previous ex vivo spectroscopic data from tissue samples revealed differences in phospholipid metabolites between isocitrate dehydrogenase mutated (IDHmut) and IDH wildtype (IDHwt) gliomas. We investigated whether these changes can be found in vivo using 1 H-decoupled 31 P magnetic resonance spectroscopic imaging (MRSI) with 3D chemical shift imaging (CSI) at 3 T in patients with low and high-grade gliomas., Methods: The study included 33 prospectively enrolled, mostly untreated patients who met spectral quality criteria according to the World Health Organization (WHO II n = 7, WHO III n = 17, WHO IV n = 9; 25 patients IDHmut, 8 patients IDHwt). The MRSI protocol included 1 H decoupled 31 P MRSI with 3D CSI (3D 31 P CSI), 2D 1 H CSI and a  1 H single voxel spectroscopy sequence (TE 30 ms) from the tumor area. For 1 H MRS, absolute metabolite concentration values were calculated (phantom replacement method). For 31 P MRS, metabolite intensity ratios were calculated for the choline (C) and ethanolamine (E)-containing metabolites., Results: In our patient cohort we did not find significant differences for the ratio of phosphocholine (PC) and phosphoethanolamine (PE), PC/PE, (p = 0.24) for IDHmut compared to IDHwt gliomas. Furthermore, we found no elevated ratios of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), GPC/GPE, (p = 0.68) or GPC/PE (p = 0.12) for IDHmut gliomas. Even the ratio (PC+GPC)/(PE+GPE) showed no significant differences with respect to mutation status (p = 0.16). Nonetheless, changes related to tumor grade regarding intracellular pH (pH i ) and phospholipid metabolism as well as absolute metabolite concentrations of co-registered 2D 1 H CSI data for tumor and control tissue showed the anticipated results., Conclusion: Using 3D-CSI data acquisition, in vivo 31 P MR spectroscopic measurement of phospholipid metabolites could not distinguish between IDHmut and IDHwt.

Study Question: Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa?, Summary Answer: Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface., What Is Known Already: Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm., Study Design, Samples/materials, Methods: Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm., Main Results and the Role of Chance: DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87., Limitations, Reasons for Caution: Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm., Wider Implications of the Findings: 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility., Study Funding and Competing Interest(s): This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.)

A method was developed for analyzing broad spectrum small molecule metabolites in the serum of hepatocellular carcinoma (HCC) patients based on ultrafast liquid chromatography-ion trap-time of flight tandem mass spectrometry (UFLC-IT-TOF MS). Serum samples were collected from 80 HCC patients and healthy persons. After pretreatment process for protein precipitation, the supernatant was analyzed with the UFLC-IT-TOF MS to obtain information on the metabonomics of small molecules. The eight compounds of glycocholic acid, choline glycerophosphate, acetyl-L-phenylalanine, oleamide, tetradecanamide, acetylcarnitine, lysolecithin and glycochenodeoxycholic acid in the HCC group were identified with significant differences from those in the health group (P <0.01). By using multidimensional analysis of variation coefficient and principal component analysis for the repeatability and 48 h stability, the method was demonstrated to have good repeatability, excellent precision, and high stability, which can satisfy the metabonomics research requirement. The high throughput and practical usability of the method further shows perspective for metabonomic analysis of large-batch serum samples.

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high-energy electrons (>20 eV) to induce gas-phase dissociation of intact precursor ions results in diagnostic product ions for localizing double-bond positions and determining acyl chain assignment. We describe a high-resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron-induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double-bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron-based dissociation technique with high-resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation., (Copyright © 2015 John Wiley & Sons, Ltd.)

Lipidomic analysis of the complex mixture of lipids isolated from biological systems can be a challenging process that often involves tandem mass spectrometry and interpretation of both precursor ions and product ions relative to the molecular structure of the lipids. Therefore, detailed understanding of the gas-phase ion chemistry occurring for each class of phospholipids is critically important for an accurate assignment of lipid structure. Some oxidized phosphatidylcholines are known to be biologically active and responsible for pathological events, and are therefore important targets for detection in lipidomic studies. Modification of fatty acyl chains by oxidation may, however, change the behavior of ion formation and decomposition in the mass spectrometer. In this study, we report on the mass-spectrometric behavior of 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, a bioactive product of phosphatidylcholine oxidation. In addition to [M-15](-) and the acetate adduct [M+59](-), three additional adduct ions, including [M-H](-), were present in significant abundance in the negative ion electrospray mass spectrum. It was found that this unexpected [M-H](-) ion was formed by the transfer of a methyl group from the choline residue on the polar head group to the aldehyde functionality of the sn-2 substituent, resulting in a 14-Da increase in the mass of the resulting sn-2 carboxylate anion formed by collisional activation of this ion. These results suggest additional rules for understanding the gas-phase ion chemistry of aldehydic phosphatidylcholine molecular species.

Becker muscular dystrophy (BMD) is characterized by progressive muscle weakness. Muscles show structural changes (fatty infiltration, fibrosis) and metabolic changes, both of which can be assessed using MRI and MRS. It is unknown at what stage of the disease process metabolic changes arise and how this might vary for different metabolites. In this study we assessed metabolic changes in skeletal muscles of Becker patients, both with and without fatty infiltration, quantified via Dixon MRI and (31) P MRS. MRI and (31) P MRS scans were obtained from 25 Becker patients and 14 healthy controls using a 7 T MR scanner. Five lower-leg muscles were individually assessed for fat and muscle metabolite levels. In the peroneus, soleus and anterior tibialis muscles with non-increased fat levels, PDE/ATP ratios were higher (P < 0.02) compared with controls, whereas in all muscles with increased fat levels PDE/ATP ratios were higher compared with healthy controls (P ≤ 0.05). The Pi /ATP ratio in the peroneus muscles was higher in muscles with increased fat fractions (P = 0.005), and the PCr/ATP ratio was lower in the anterior tibialis muscles with increased fat fractions (P = 0.005). There were no other significant changes in metabolites, but an increase in tissue pH was found in all muscles of the total group of BMD patients in comparison with healthy controls (P < 0.05). These findings suggest that (31) P MRS can be used to detect early changes in individual muscles of BMD patients, which are present before the onset of fatty infiltration., (Copyright © 2014 John Wiley & Sons, Ltd.)

Here, we show that the sensitivity of (31)P MRSI of (31)P spins J-coupled to protons can be increased by almost a factor of three when compared with an optimal direct detection free induction decay. By direct detection integrated with multi-echo polarization transfer (DIMEPT), multiple signals from polarization transfer and direct detection can be acquired in one repetition time, with minimal mutual interference, provided that the number of refocusing pulses in the multi-echo polarization transfer part is even. The DIMEPT sequence was implemented on a 7-T body scanner and tested on a phantom and on the breasts of five healthy volunteers. The in vivo signal-to-noise ratio (SNR) enhancement for the J-coupled phosphomonoesters was 270% when compared with an Ernst angle pulse-acquire sequence. However, the phosphodiester signals, presumably mainly mobile phospholipids, had T2 values that were too short to be enhanced. Uncoupled (31)P spins, with sufficiently long T2 values, such as inorganic phosphate, were SNR enhanced by a factor of 1.9 relative to an Ernst-angle excitation pulse-acquire sequence by multi-echo direct detection., (Copyright © 2014 John Wiley & Sons, Ltd.)

Background: Prenatal alcohol exposure has been linked to impairment in cerebellar structure and function, including eyeblink conditioning. The deep cerebellar nuclei, which play a critical role in cerebellar-mediated learning, receive extensive inputs from brain stem and cerebellar cortex and provide the point of origin for most of the output fibers to other regions of the brain. We used in vivo (1) H magnetic resonance spectroscopy (MRS) to examine effects of prenatal alcohol exposure on neurochemistry in this important cerebellar region., Methods: MRS data from the deep cerebellar nuclei were acquired from 37 children with heavy prenatal alcohol exposure and 17 non- or minimally exposed controls from the Cape Coloured (mixed ancestry) community in Cape Town, South Africa., Results: Increased maternal alcohol consumption around time of conception was associated with lower N-Acetylaspartate (NAA) levels in the deep nuclei (r = -0.33, p < 0.05). Higher levels of alcohol consumption during pregnancy were related to lower levels of the choline-containing metabolites (r = -0.37, p < 0.01), glycerophosphocholine plus phosphocholine (Cho). Alcohol consumption levels both at conception (r = 0.35, p < 0.01) and during pregnancy (r = 0.38, p < 0.01) were related to higher levels of glutamate plus glutamine (Glx). All these effects continued to be significant after controlling for potential confounders., Conclusions: The lower NAA levels seen in relation to prenatal alcohol exposure may reflect impaired neuronal integrity in the deep cerebellar nuclei. Our finding of lower Cho points to disrupted Cho metabolism of membrane phospholipids, reflecting altered neuropil development with potentially reduced content of dendrites and synapses. The alcohol-related alterations in Glx may suggest a disruption of the glutamate-glutamine cycling involved in glutamatergic excitatory neurotransmission., (Copyright © 2014 by the Research Society on Alcoholism.)

Huntington's disease (HD) is a neurologic disorder that is not completely understood; its fundamental physiological mechanisms and chemical effects remain somewhat unclear. Among these uncertainties, we can highlight information about the concentrations of brain metabolites, which have been widely discussed. Concentration differences in affected, compared to healthy, individuals could lead to the development of useful tools for evaluating the progression of disease, or to the advance of investigations of different/alternative treatments. The aim of this study was to compare the thalamic concentration of metabolites in HD patients and healthy individuals using magnetic resonance spectroscopy. We used a 2.0-Tesla magnetic field, repetition time of 1500 ms, and echo time of 135 ms. Spectra from 40 adult HD patients and 26 control subjects were compared. Quantitative analysis was performed using the LCModel method. There were statistically significant differences between HD patients and controls in the concentrations of N-acetylaspartate+N-acetylaspartylglutamate (NAA+NAAG; t-test, P<0.001), and glycerophosphocholine+phosphocholine (GPC+PCh; t-test, P=0.001) relative to creatine+phosphocreatine (Cr+PCr). The NAA+NAAG/Cr+PCr ratio was decreased by 9% and GPC+PCh/Cr+PCr increased by 17% in patients compared with controls. There were no correlations between the concentration ratios and clinical features. Although these results could be caused by T1 and T2 changes, rather than variations in metabolite concentrations given the short repetition time and long echo time values used, our findings point to thalamic dysfunction, corroborating prior evidence.

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many "targeted" approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Background: Endogenous phospholipids have a profound matrix effect in bioanalytical LC-MS methods and considerable effort is invested in strategies to minimize their impact either by removal during sample processing or chromatographic separation during the analytical run. The aim of the research presented in this article was to investigate the latter approach, under reversed-phase conditions., Results: The retention of glycerophosphocholines (GPCs) in mobile phases employing acetonitrile demonstrated a complex 'U-shaped' relationship with the percentage of organic. Conversely, in mobile phases employing methanol, the relationship between retention and percentage of organic was entirely predictive and unaffected by changes in the mobile phase pH. The GPC elution profile was also qualitatively equivalent irrespective of the species from which the plasma was derived., Conclusion: The predictive nature of GPC retention, under reversed-phase chromatography and with MeOH as organic modifier, is an important finding that should allow for a more streamlined and simplified strategy in the development of bioanalytical assays.

Background: Suppression or enhancement of MS ionization, particularly evident when electrospray is used as the source of ions, has been widely discussed., Methods: An assay for a small-molecule pharmaceutical in dog plasma between 1-300 ng/ml was validated with a mean bias across the calibration range of 5.0%. When the calibration sample matrix was substituted for human plasma, the mean bias across the range increased to 29.1%. A study of bias originating as a result of matrix effects, arising from endogenous glycerophosphocholine species, in plasma sources is discussed., Conclusion: A simple strategy to assess the potential of any unmitigated matrix effect to bias quantitative analysis by nonequivalent ionization induction or suppression is evaluated.

Thin stillage contains organic and inorganic compounds, some of which may be valuable fermentation coproducts. This study describes a thorough analysis of the major solutes present in thin stillage as revealed by NMR and HPLC. The concentration of charged and neutral organic compounds in thin stillage was determined by excitation sculpting NMR methods (double pulse field gradient spin echo). Compounds identified by NMR included isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol, and 2-phenylethanol. The concentrations of lactic and acetic acid determined with NMR were comparable to those determined using HPLC. HPLC and NMR were complementary, as more compounds were identified using both methods. NMR analysis revealed that stillage contained the nitrogenous organic compounds betaine and glycerophosphorylcholine, which contributed as much as 24% of the nitrogen present in the stillage. These compounds were not observed by HPLC analysis.

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers., (Copyright © 2010 AACR.)

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.

In this research, our aim was to isolate and characterize the substance known as "meat factor," which is reported to enhance nonheme iron absorption. We used various analytical techniques, and the final step was a human study to measure the effect of a candidate compound on iron absorption. Lean beef was selected for study, as it is known to increase nonheme iron absorption. Cooked ground beef was homogenized and aliquots were taken through a simulated gastric and intestinal digestion. This was followed by purification using fast protein liquid chromatography. The fractions were collected and applied to a Caco-2 cell system designed to measure iron absorption using radioiron. Fractions with an enhancing effect were analyzed by mass spectrometry, nuclear magnetic resonance, and HPLC, and a proposed empirical formula was obtained for the substance in the most active fraction (C(8)H(20) NO(6)P). Tandem mass spectrometry was used to identify the compound as L-alpha-glycerophosphocholine (L-alpha) by comparing the spectra against authentic material. We added a commercially available food grade source of L-alpha to vegetarian lasagna, with and without 100 mg ascorbic acid (a known enhancer of nonheme iron absorption), at the same enhancer:iron molar ratio (2:1), and fed meals to 13 women of child-bearing age with low iron stores. The nonheme iron was labeled with stable isotopes of iron to provide a total dose per meal of 10 mg iron, and absorption was measured from erythrocyte incorporation. Nonheme iron absorption from lasagna was increased by the addition of either ascorbic acid (P = 0.010) or L-alpha (P = 0.023). We have identified L-alpha as a component of muscle tissue that enhances nonheme iron absorption, and this finding provides new opportunities for iron fortification of foods.

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduct ion (e.g., [M - H](-), [M - 2H](2-), [M + H](+), [M + Na](+), [M + NH(4)](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

Hypoinsulinemia characteristic to type 1 diabetes may theoretically inhibit the conversion of essential fatty acids to their longer-chain metabolites. Fatty acids were determined in plasma and erythrocyte membrane lipids in young diabetic adults (n=34) and in age-matched healthy controls (n=36). Values of linoleic acid (56.01 [5.02] versus 51.05 [7.32], % by wt, median [range from the first to the third quartile], P<0.00l) and arachidonic acid (AA) (11.17 [2.98] versus 9.69 [1.95] P<0.001) were significantly higher in diabetic subjects than in controls. However, alpha-linolenic acid values did not differ, and docosahexaenoic acid (0.43 [0.12] versus 0.57 [0.29], P<0.01) values were significantly lower in diabetic than in control subjects. Significant inverse correlations were found between AA and hemoglobin A(1c) values in the phospholipid (r=-0.40, P<0.05) and sterol ester (r=-0.40, P<0.05) fractions. The data obtained in the present study suggest that the availability of n-3 long-chain polyunsaturated fatty acid may be reduced in young diabetic adults.

Elevated skeletal muscle phosphodiesters (PDE) have previously been reported with muscle-related disorders. Myalgia is a side effect of using statin cholesterol-lowering medications and, therefore, statin use may be associated with increased skeletal muscle PDE. The effect of cholesterol-lowering drugs on skeletal muscle phosphorus metabolites was determined with (31)P magnetic resonance spectroscopy. Resting (31)P metabolites of the anterior compartment muscles were measured in two groups (n = 20; age, 49 +/- 2 years); half were taking statins and the other half were not on these agents. Muscle PDE was 57% greater in the statin group than the control group. These data suggest that statin use increases muscle PDE. Our findings are particularly relevant due to the increasing use and higher dosing of statin medications. Further prospective studies should be performed to document a causal relationship between elevated PDE and statin use, in addition to quantifying correlates to muscle function.

Invertebrates at hydrothermal vents and cold seeps must cope with high levels of toxic H2S. In addition, these and all marine invertebrates must balance internal osmotic pressure with that of the ocean. Cells usually do so with organic osmolytes, primarily free amino acids (e.g., taurine, glycine) and methylamines (e.g., betaine). At vents and seeps, clams, mussels, and vestimentiferans with thiotrophic endosymbionts have high levels of hypotaurine and thiotaurine (a product of hypotaurine and HS-). These serve as osmolytes but their primary function may be to transport and/or detoxify sulfide; indeed, thiotaurine has been proposed to be a marker of thiotrophic symbiosis. To test this, we analyzed Depressigyra globulus snails and Lepetodrilus fucensis limpets from Juan de Fuca Ridge vents (1,530 m). Neither has endosymbionts, though the latter has thiotrophic ectosymbionts. Some specimens were rapidly frozen, while other live ones were kept in laboratory chambers, some with and others without sulfide. Non-vent gastropods from a variety of depths (2-3,000 m) were also collected. Tissues were analyzed for major osmolytes and taurine derivatives. The dominant osmolytes of non-vent snails were betaine in all species, and either taurine in shallow-living species or scyllo-inositol, glycerophosphorylcholine, and other amino acids in deep-sea species. In contrast, the dominant osmolytes were hypotaurine and betaine in D. globulus, and hypotaurine in L. fucensis. Both species had thiotaurine (as well as hypotaurine) at levels much greater than previously reported for vent and seep animals without endosymbionts. The ratio of thio- to thio- plus hypotaurine, a possible indicator of sulfide exposure, decreased in both species when kept in laboratory chambers with low or no sulfide, but stayed at high levels in snails kept with 3-5 mM sulfide. Thus, in some vent animals without endosymbionts, sulfide may be detoxified via conversion of hypotaurine to thiotaurine. The latter may be a marker of high sulfide exposure but not of thiotrophic endosymbionts., (Copyright 2006 Wiley-Liss, Inc.)

Phosphoinositide 3-kinase (PI3K) is an attractive target for novel mechanism-based anticancer treatment. We used magnetic resonance (MR) spectroscopy (MRS) to detect biomarkers of PI3K signaling inhibition in human breast cancer cells. MDA-MB-231, MCF-7, and Hs578T cells were treated with the prototype PI3K inhibitor LY294002, and the (31)P MR spectra of cell extracts were monitored. In every case, LY294002 treatment was associated with a significant decrease in phosphocholine levels by up to 2-fold (P < 0.05). In addition, a significant increase in glycerophosphocholine levels by up to 5-fold was also observed (P

Acetonitrile extracts of ulcerated and control rat stomachs were studied by various NMR techniques in an attempt to understand how indomethacin, a common and powerful nonsteroidal antiinflammatory drug (NSAID), induces ulcers in the stomach. One- (1D) and two-dimensional (2D) NMR spectra of extracts of ulcerated and control stomachs revealed that glycolytic and Krebs cycle enzymes were partially inhibited in the ulcerated stomach as shown by the lactate/glucose ratio. The (total choline)/lactate ratio was also higher in the extract from the control stomach than in the ulcerated stomach. Glycerophosphoethanolamine and glycerophosphocholine concentrations were higher in the ulcerated stomach extract as compared with the control stomach extract. These results explain the gastrointestinal protective effect of D-glucose and Krebs cycle intermediates on NSAID-induced ulceration.

Study Design: The present study was carried out to assess the metabolic differences between cerebrospinal fluid samples of patients with spina bifida and age-matched control individuals., Objectives: To study the metabolite profile of cerebrospinal fluid of patients with spina bifida using proton magnetic resonance spectroscopy, compare the levels of metabolites with controls, establish correlation of underlying neuronal dysfunction with metabolic changes in patients with spina bifida, and evaluate the potential use of this technique as an additional tool for diagnostic assessment., Summary of Background Data: Combination of embryopathy, stretching, ischemia, compression, and trauma is responsible for cord dysfunction in spina bifida. Changes in neuronal metabolism leads to changes in the local milieu of cerebrospinal fluid in the cord. Change in metabolite profile of cerebrospinal fluid in spina bifida in terms of increase in products of anaerobic metabolism, nerve membrane integrity, and nerve ischemia has not yet been studied., Methods: Cerebrospinal fluid obtained from patients and control individuals were characterized using various one- and two-dimensional proton magnetic resonance spectroscopy techniques. Concentration of various metabolites was calculated using the area under the nuclear magnetic resonance peak., Results: Statistically significantly higher levels of lactate, choline, glycerophosphocholine, acetate, and alanine in the cerebrospinal fluid of patients with spina bifida was observed compared with control individuals., Conclusions: Significantly higher levels of metabolites were observed in patients with spina bifida, representing a state of nerve ischemia, anaerobic metabolism, and disruption of neuronal membrane.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease linked to the lack of the dystrophin, a submembrane protein, leading to muscle weakness and associated with a defect of the lipid metabolism. A study of the fatty acid composition of glycerophosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and tandem mass spectrometry (MS/MS) enabled us to characterize a change of the lipid composition of dystrophic cells at the time of the differentiation. This modification has been used as a marker to identify with profiling and imaging MALDI-ToF MS regenerating areas in sections of an mdx mouse leg muscle. It is the first time that such a slight change in fatty acid composition has been observed directly on tissue slices using mass spectrometry. This approach will be useful in monitoring the treatment of muscular regeneration.

Phospholipid metabolism is tightly involved in tumor growth regulation and tumor cell survival. The response of phospholipid metabolism to chloroethyle nitrosourea treatment is investigated in a murine B16 melanoma model. Measurements of phospholipid derivatives are performed on intact tumor tissue samples using one- and two-dimensional proton NMR spectroscopy. During the tumor growth inhibition phase under treatment, tumors overexpress phosphocholine, phosphoethanolamine, glycerophosphocholine and glycerophosphoethanolamine, whereas phosphatidylcholine and phosphatidylethanolamine levels are maintained to control levels. During re-growth, which remained quantitatively much below control growth, chloroethyle nitrosourea-treated melanoma tumors overexpress phosphocholine and phosphoethanolamine only. In treated melanoma, phosphatidylcholine levels show an inverse relationship with tumor growth rates. In conclusion, chloroethyle nitrosourea-treated melanoma tumors maintain their phosphatidylcholine levels and exhibit transformed phospholipid metabolism phenotype, by mechanisms that could participate in tumor cell survival.

Positive ion tandem quadrupole mass spectrometric methods for structural characterization of the subclasses of sn-glycero-3-phosphocholine (PC), including alkylacyl- and alk-1-enylacylphosphocholine and lysophosphatidylcholine (LPC), are described. Following collisionally activated dissociation, the [M + Li](+) ions generated by electrospray ionization yield abundant informative fragment ions that permit structural determination, and distinction of regioisomers among lysophosphatidylcholine can be easily achieved. In contrast, structurally informative ions arising from [M + H](+) or [M + Na](+) ions are less prominent. The most abundant ion observed in the product-ion spectra of the [M + Li](+) ions of plasmenyl- and plasmanyl-PC and of LPC arises from loss of N(CH(3))(3) ([M + Li - 59](+)). This feature permits their distinction from a product-ion spectrum arising from a diacylphosphatidylcholine, in which the [M + Li - 183](+) ion reflecting loss of phosphocholine is the most prominent. Examples for identification of various subclasses of PC in biological extracts by tandem mass spectrometry applying various constant neutral loss scannings are also shown., (Copyright 2003 John Wiley & Sons, Ltd.)

Complexes of starch and monoacyl-sn-glycerophosphatidylcholine (GPC) containing various acyl (myristoyl, palmitoyl, and stearoyl) chains were subjected to hydrolysis with glucoamylase (EC 3.2.1.3). The enzyme hydrolyzed approximately 40% of starch control and 20-28% of starch-GPC complexes. Among the GPCs examined, 1- and 2-monomyristoyl-sn-GPC showed the highest resistance to enzyme hydrolysis, and the hydrolysis rate of starch-GPCs was greater with longer chains. Enzymatic hydrolysis strongly affected the thermal properties of the starch. After enzymatic hydrolysis of starch-GPC complexes for 24 h, their thermograms had broader peaks with lower enthalpies than the corresponding starch without enzyme; however, the starch-GPC complexes showed little change. The surface of starch-GPC granules was less eroded. These results showed that the increasing amount of starch-GPC complexes could be more resistant to hydrolysis.

Background: The biochemical composition of human neoplastic and normal renal tissues., Materials and Methods: Thirteen patients with nephrocarcinomas were examined: 24 samples, 13 from the nephrocarcinomas, 9 from the surrounding healthy parenchyma and 2 from the healthy cortex and medulla were extracted and analyzed by Magnetic Resonance Spectroscopy (MRS) and High Performance Liquid Chromatography (HPLC)., Results: MRS yielded information on renal osmolytes, whereas HPLC disclosed the amino acid pattern of tissue extracts. Significant biochemical differences were found between normal and pathological renal tissues: the osmolyte content decreases dramatically in nephrocarcinomas., Conclusion: We confirm that many osmolytes are present in the healthy kidney, with a different distribution between cortex and medulla, and can be considered markers of a physiological renal function. The marked decrease of these osmolytes is typical of cancer. A detailed knowledge of the biochemical composition of human renal tissues is required for the use of diagnostic methods, like in vivo MRS, the reliability of which is based on the detection of molecular markers. Useful information can also be obtained from the study of the amino acidic fraction.

When collisionally activated dissociation (CAD) of glycerophosphocholine (GPC) species is examined using quadrupole ion trap mass spectrometry (QITMS), the spectral patterns differ from those obtained using sector or quadrupole mass spectrometry. Methods employed in the structural analysis of GPCs using a sector or quadrupole mass spectrometers are not necessarily useful for an ion trap mass spectrometer. A novel method is presented for structurally analyzing GPCs that involves the CAD of trifluoroacetic acid (TFA) adducts of kaliated GPCs. Solutions of GPCs in 0.1% TFA/methanol were electrosprayed to produce precursor ions by attaching a trifluoroacetic acid (TFA) molecule to a kaliated GPC molecule. The CAD-MS/MS spectra obtained by QITMS revealed a dramatic increase in the abundance of fragment ions, corresponding to the losses of sn-1 and sn-2 fatty acyl substituents. A preferential loss of the sn-1 fatty acyl group over the loss of the sn-2 fatty acyl group was observed among the GPC standards examined. A GPC extract from egg yolk was directly analyzed by this method without prior separation. The identities and positions of fatty acyl substituents of over 20 GPC species were identified. Some isomers present in very low relative abundance, which could not be analyzed by QITMS/MS using other ions as precursors, were identified by the TFA attachment method., (Copyright 2002 John Wiley & Sons, Ltd.)

Platelet-activating factor (PAF) is an early product of the inflammatory environment, influencing development and resolution of inflammation. Its production is greater in neutrophils and macrophages, which predominantly synthesize 1-alkyl sn-2 acetyl glycerophosphocholine (GPC) than in nongranulocytes (B cells and endothelial cells), which lack a respiratory burst and synthesize 1-acyl sn-2 acetyl GPC as their major PAF species. This study investigated whether the respiratory burst was responsible for the quantitative and qualitative differences in sn-2 acetyl GPC species generation by neutrophils and macrophages versus those cells lacking the NADPH oxidase complex. The myeloid cell line PLB-985 (capable of differentiation into neutrophils) was used to test this hypothesis, since these cells had previously been generated with a non-functional respiratory burst (X-CGD PLB-985). Differentiated PLB-985 cells underwent a large respiratory burst in response to PMA (phorbol ester), and smaller respiratory bursts in response to A23187 (calcium ionophore), and the bacterial polypeptide fMLP (receptor mediated activation). Concurrently, treated cells were assessed for production of 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC species by gas chromatography/mass spectrometry. Neither cell type generated these lipid species in response to PMA, but both cell types generated equal levels of sn-2 acetyl GPC in response to A23187, with five times more 1-hexadecyl than 1-palmitoyl species. Upon fMLP activation, X-CGD PLB-985 cells produced significantly less 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC in comparison to the wild-type PLB-985 cells. These findings suggest phagocytic oxidant production by NADPH oxidase is not essential for sn-2 acetyl GPC generation, but appears important for optimal production of PAF in response to some stimuli.

Introduction: To measure the levels of phosphocholine (PCh) and glycerophosphocholine (GPCh) in the tissues and organs of mice, we developed a simple and rapid method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column., Methods: Under our modifications of the separation procedure of Klein et al. [Neurochem. Int. 2 (1993) 293], PCh and GPCh in the hydrophilic phase of the homogenate samples were selectively hydrolyzed into free choline by alkaline phosphatase and a 0.4-N perchloric acid solution, respectively, and the resulting hydrolyzed mixtures were directly injected into the HPLC system for analysis., Results: The present method permits PCh or GPCh assay within 5 min in one chromatographic run. Recoveries from tissue samples were 97% for PCh and 101% for GPCh. The percentages of the crossover reaction to the authentic PCh and GPCh were 0.4% and 3.8%, respectively. The within-run coefficients of variation for choline derived from PCh and GPCh in the tissue samples were 1.2% and 1.4%, respectively., Discussion: The method is effective and has been applied to the measurement of PCh and GPCh levels in several tissues of mice.

Nuclear magnetic resonance (NMR) spectroscopy was used to quantify citrate, glucose, lactate, glycerophosphorylcholine and choline in seminal plasma from subjects injected with a new male contraceptive RISUG, a copolymer of styrene maleic anhydride dissolved in dimethyl sulphoxide, and in seminal plasma from normal ejaculates. No significant difference in the concentration of citrate was observed between the groups, indicating that the prostate is not affected by the contraceptive. The concentrations of glucose, lactate, glycerophosphorylcholine and choline were significantly lower (P < 0.01) in subjects injected with RISUG compared with controls. In addition, metabolite ratios such as choline:citrate, citrate:lactate, choline:lactate and glycerophosphorylcholine:choline were calculated. Citrate:lactate and glycerophosphorylcholine:choline ratios were significantly lower in RISUG-injected subjects than in controls (P < 0.01), thereby indicating the occurrence of partial obstructive azoospermia. The most important finding of the present study was that the intervention of RISUG in the vas deferens even for a period as long as 8 years is absolutely safe and does not lead to prostatic diseases.

Earlier studies have shown that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) and the photostabilizer 2-hydroxy-4-methoxybenzophenone (HMBP) are cytotoxic and inhibit cell growth. It was the aim of this study to elucidate the underlying metabolic effects of TEGDMA and HMBP on immortal contact-inhibited Swiss albino mouse embryo cells (3T3 fibroblasts) by nuclear magnetic resonance (NMR) spectroscopy. Cell extracts and culture media were analyzed by NMR spectroscopy for metabolic changes after incubation for 24 hours with ED20-concentrations of TEGDMA and HMBP. TEGDMA could be detected in all fractions (cytosol, lipid fractions, and culture media) of 3T3 cells, while HMBP was found only in the lipid fraction accumulated at a maximum rate (51 nmol/mg DNA) compared with TEGDMA (27 nmol/mg DNA). TEGDMA increased the concentration of phosphomonoesters to 180+/-36% and decreased the phosphodiesters to 65+/-5% of controls (control = 100%). Thus, the turnover of phospholipids was enhanced, whereas content and composition of phospholipids of membranes did not alter markedly. Additionally, TEGDMA changed the metabolic state of cells, indicated by slight decreases of nucleoside triphosphates and an increase in the ratio of nucleoside diphosphates to nucleoside triphosphates, while HMBP had no effect. The most remarkable effect of TEGDMA was a nearly complete decline of the intracellular glutathione levels. Analysis of our data shows that NMR spectroscopy of cell-material interactions may reveal metabolic effects of organic test substances which are not detectable by standard in vitro assays. The comonomer TEGDMA affected the metabolism of the cells on different levels, while HMBP accumulated in the lipid fraction and induced significantly fewer effects on cell metabolism.

Unlabelled: Choline is an essential constituent of membrane phospholipids in great demand in the developing brain and liver. We have previously demonstrated that human neonates excrete large amounts of betaine, a product of choline oxidation, in their urine. Estimates of perinatal choline intake using previously published data compared with our measurements of betaine excretion indicated that choline insufficiency might occur at certain periods after birth. In this study we measured the choline content of expressed human breast milk in colostrum (2-6 days after birth), mature milk (7-22 days after birth) and several infant formula foods, using proton nuclear magnetic resonance spectroscopy. In colostrum choline was present in both the aqueous (free choline, phosphocholine and glycerophosphocholine) and lipid fractions (phosphatidylcholine and sphingomyelin). After 6-7 days there was a mean increase of 114% in the total choline content; 82% of the rise was accounted for by increases in phosphocholine and glycerophosphocholine, and 14% by (free) choline. The choline content of most formula feeds was comparable with the level in colostrum but below that of mature milk. Both the total choline content and ester composition of mature milk were comparable with more recent measurements using high-performance liquid chromatography., Conclusion: The choline content of human breast milk doubles 6-7 days after birth and, unlike that of many formula feeds, appears to be sufficient to account for betaine excretion in healthy full-term neonates. However, for premature babies who usually receive much lower quantities of milk, yet have a higher demand for choline, the intake may be inadequate.

The renal concentrating defect typical for chronic K depletion has been ascribed to malfunction of renomedullary cells caused by inadequate accumulation of organic osmolytes. A reduction in intracellular ionic strength, which is believed to influence decisively the accumulation of organic osmolytes, has been held responsible for insufficient osmolyte accumulation. To test this hypothesis, intra- and extracellular Na, Cl and K concentrations, the major determinants of ionic strength, were measured in the papilla by electron microprobe analysis and organic osmolytes (glycerophosphorylcholine, betaine, sorbitol, myo-inositol, free amino acids) in inner-medullary tissue by HPLC in antidiuretic rats kept on either a control (normal-K) or a K-deplete (low-K) diet and in euhydrated rats with free access to water and control diet. K depletion was associated with a reduced urine concentrating ability. Papillary interstitial ionic strength (sum of Na, Cl and K) in antidiuretic low-K rats was significantly reduced compared with antidiuretic normal-K rats (688+/-19 vs. 971+/-61 mmol/kg wet wt) but was similar to that in euhydrated normal-K rats (643+/-35 mmol/kg wet wt). The lower interstitial ionic strength in antidiuretic low-K and euhydrated normal-K rats was associated with a lower total content of organic osmolytes in the inner medulla (365+/-14 and 381+/-20, respectively, vs. 465+/-11 mmol/kg protein in antidiuretic normal-K rats). Intracellular ionic strength (sum of Na, Cl and K) of papillary collecting duct cells, however, was similar in antidiuretic normal-K and euhydrated normal-K rats (171+/-5 and 179+/-11 mmol/kg wet wt) but lower in antidiuretic low-K rats (138+/-9 mmol/kg wet wt). These results do not support the view that, in the steady state of osmotic adaptation of renomedullary cells in situ, intracellular ionic strength is the decisive factor for maintaining high levels of organic osmolytes. During chronic K depletion, reduced osmolyte accumulation by renomedullary cells may be the consequence, rather than the cause, of lower medullary interstitial tonicity.

Oxidized low-density lipoprotein (LDL) is thought to play an important role in atherogenesis and cardiovascular disease in humans. Oxidized LDL is a complex mixture of many oxidized species, including numerous oxidized glycerophospholipids. Electrospray ionization and tandem mass spectrometry as well as microchemical derivatization of high-performance liquid chromatographically purified fractions derived from oxidized LDL were investigated as means to determine the structure of individual components present in oxidized LDL. One major oxidized phosphocholine lipid had an [M + H](+) ion at m/z 650. Derivatization to the trimethylsilyl ether and methoxime caused shifts in mass which, along with negative ion collision-induced dissociation mass spectra, were consistent with the presence of three species, 1-palmitoyl-2-(9-oxononanoyl)glycerophosphocholine and two isomeric 1-octadecanoyl-2-(hydroxyheptenoyl)glycerophosphocholines. These species were chemically synthesized. Trimethylsilylation of free hydroxyl groups increased the mass of the phospholipid acyl chains containing hydroxyl groups by 72 u. Conversion of carbonyl groups to the methoxylamine derivative increased the mass by 29 u. Ozonolysis of those products which contained double bonds proved to be a facile technique to determine the position and number of double bonds present. The use of these techniques was illustrated in the structural characterization of one major component (m/z 650, positive ions) in oxidized LDL as 1-octadecanoyl-2-(7-hydroxyhepta-5-enoyl)glycerophosphocholi ne. A possible mechanism for the formation of this unique chain-shortened glycerophospholipid is proposed., (Copyright 2000 John Wiley & Sons, Ltd.)

Nm23 genes are involved in the control of the metastatic potential of breast carcinoma cells. To understand the impact of nm23 genes on tumor physiology and metabolism, a 31P nuclear magnetic resonance (NMR) spectroscopic study was performed on tumors formed in the mammary fat pad of severe combined immunodeficiency mice by MDA-MB-435 human breast carcinoma cells transfected with cDNA encoding wild type nm23-H1 and nm23-H2 proteins. Tumors formed by MDA-MB-435 cells transfected with vector alone were used as controls. All transgene tumors exhibited significantly higher levels of phosphodiester (PDE) compounds relative to phosphomonoester (PME) compounds in vivo compared with control tumors. Similar differences in PDE and PME also were observed for spectra obtained from cells growing in culture. Intracellular pH was significantly lower and extracellular pH was significantly higher for transgene tumors compared with control tumors. Histologic analysis of lung sections confirmed reductions in incidence, number, and size of metastatic nodules for animals bearing transgene tumors. These results suggest that nm23 genes may affect suppression of metastasis through phospholipid-mediated signaling and cellular pH regulation.

Through its pro-inflammatory effects on leukocytes, endothelial cells, and keratinocytes, the lipid mediator platelet-activating factor (PAF) has been implicated in cutaneous inflammation. Although the 1-alkyl PAF species has been considered historically the most abundant and important ligand for the PAF receptor (PAF-R), other putative ligands for this receptor have been described including 1-acyl analogs of sn-2 acetyl glycerophosphocholines. Previous bioassays have demonstrated a PAF-like activity in lesions of the autoimmune blistering disease bullous pemphigoid. To assess the actual sn-2 acetyl glycerophosphocholine species that result in this PAF agonistic activity, we measured PAF and related sn-2 acetyl GPCs in fresh blister fluid samples from bullous pemphigoid and noninflammatory (suction-induced) bullae by mass spectrometry. We report the presence of 1-hexadecyl as well as the 1-acyl PAF analog 1-palmitoyl-2-acetyl glycerophosphocholine (PAPC) in inflammatory blister fluid samples. Because PAPC is the most abundant sn-2 acetyl glycerophosphocholine species found in all samples examined, the pharmacological effects of this species with respect to the PAF-R were determined using a model system created by transduction of a PAF-R-negative epidermoid cell line with the PAF-R. Radioligand binding and intracellular calcium mobilization studies indicated that PAPC is approximately 100x less potent than PAF. Though a weak agonist, PAPC could induce PAF biosynthesis and PAF-R desensitization. Finally, intradermal injections of PAF and PAPC into the ventral ears of rats demonstrated that PAPC was 100x less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous diseases.

Recent studies have indicated that estrogen has a stimulatory influence on the male reproductive tract. Evidence includes the presence of measurable levels of estrogen in the circulation, retention of exogenous estrogen, and presence of estrogen receptors in the male accessory sex organs during prepubertal life. In the present study, estrogen antagonists (CDRI-85/287 and centchroman) have been used to examine this concept by antagonising estrogen action at critical stages in the life in rat. Centchroman or 85/287 administration to 14 day old rats for 7 days did not alter gonadal or accessory organ weight. In contrast, in 21 day old castrated rats, treatment with either compound from day 28-35 of life stimulated an increase in all organ weights. When administered to normal rats during the critical phase of transition, i.e. days 30-60 of life, both testis and accessory organs showed an increase in weight. In contrast castrated rats treated with estrogen alone or in combination with 85/287 from days 37-45 of life and sacrificed on day 46 did not show any change, but 85/287 per se markedly reduced the weight of accessory organs. In adult castrated rats, the potency of DHT as a promoter of growth was potentiated by estradiol. Compound 85/287 negated the estradiol-induced increase. Glycerylphosphorylcholine (GPC) and sialic acid levels showed about 100% increase, with both high and low doses of 85/287 (treated from 30-60 days of life), However, centchroman (CDRI-67/20) was less potent in this regard. The effect of estrogen antagonists in relation to epididymal physiology during different phases of life in the rat is discussed.

The cell volume is regulated not only by inorganic ions, but also by organic osmolytes, such as amino acids, methylamines, and polyhydric alcohols (polyols). Using proton nuclear magnetic resonance spectroscopy (1H-NMR), we measured the tissue concentrations of amino acids (alanine, aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, N-acetyl-aspartate (NAA), taurine), methylamines (glycerophosphorylcholine (GPC), creatine+phosphocreatine (total creatine, tCr)), and polyols (myo-inositol) in the rat brain after middle cerebral artery occlusion (incomplete focal ischemia) or after decapitation (complete global ischemia). The total osmolytes expressed as a sum of total amino acids, total methylamines, and total polyols were significantly decreased at 24 h of focal ischemia (58.7% of control value, P=0.0025) whereas they were not changed following decapitation. The water content was increased from control value of 77.9%-84.1% after focal ischemia (P<0.0001) but not after decapitation. These results suggest that the brain organic osmolytes are involved in the process of edema formation following focal cerebral ischemia. Further elucidation of the cellular mechanisms regulating these organic osmolytes in cerebral ischemia may promote greater understanding of the pathophysiology involved in the evolution of brain edema.

To assess testicular function in experimental varicocele rats, we used 31P magnetic resonance (MR) spectroscopy and compared MR spectroscopic parameters with flow cytometric DNA analysis. In vivo 31P MR spectroscopy and flow cytometric DNA analysis of testes were performed in 10 sham-operated and 9 induced varicocele rats. Although the testicular phosphomonoester (PM)/ATP ratio did not differ between sham-operated and induced varicocele rats, the phosphodiester (PD)/ATP ratio was significantly reduced and the inorganic phosphate (Pi)/ATP ratio was significantly increased in induced varicocele rats. There was no difference between the right and left sides. DNA flow cytometry showed a decrease in the percentage of haploid cells in induced varicocele rats, regardless of the side. This study indicates that in vivo 31P MR spectroscopy provides valuable information for assessment of testicular function.

Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5 x 10(-7) M and 1 x 10(-6) M respectively with r.s.d. < 2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months.

Rationale and Objectives: To evaluate whether decoupling improves signal-to-noise ratio and frequency resolution of in vivo kidney spectra, and to compare native and well-functioning transplant kidneys., Methods: Proton decoupling in conjunction with three-dimensional chemical shift imaging (3D-CSI) in phosphorus-31 magnetic resonance (MR) spectroscopy was used with a spatial resolution of 64 cm3 and 17-minute acquisition time to compare native (n = 10) and well-functioning transplant (n = 9) kidneys., Results: Proton decoupling improved peak amplitudes by almost 30%, as well as chemical shift resolution of in vivo kidney spectra. No statistically significant differences in phosphometabolite ratios and renal spectra were observed between healthy volunteers and patients with nonrejecting transplants. The phosphodiester-phosphomonoester ratio was 3.02 +/- 0.88, phosphomonoester-inorganic phosphate ratio was 1.07 +/- 0.44, and inorganic phosphate-adenosine triphosphate ratio was 0.58 +/- 0.22 after correction for saturation effects., Conclusion: Improved spectra of native and transplant kidneys can be obtained in vivo with MR spectroscopy by using a short acquisition time.

Large amounts of choline are required in neonates for rapid organ growth and membrane biosynthesis. Human infants derive much of their choline from milk. In our study, mature human milk contained more phosphocholine and glycerophosphocholine than choline, phosphatidylcholine, or sphingomyelin (P < 0.01). Previous studies have not recognized that phosphocholine and glycerophosphocholine exist in human milk. Concentrations of choline compounds in mature milk of mothers giving birth to preterm or full-term infants were not significantly different. Infant formulas also contained choline and choline-containing compounds. In infant formulas derived from soy or bovine milk, unesterified choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin concentrations varied greatly. All infant formulas contained significantly less phosphocholine than did human milk. Soy-derived formulas contained significantly less glycerophosphocholine (P < 0.01) and phosphocholine (P < 0.01) and more phosphatidylcholine (P < 0.01) than did human or bovine milk or bovine milk-derived infant formulas. Rat milk contained greater amounts of glycerophosphocholine (almost 75% of the total choline moiety in milk) and phosphocholine than did human milk. When dams were provided with either a control, choline-deficient, or choline-supplemented diet, milk composition reflected the choline content of the diet. Because there are competing demands for choline in neonates, it is important to ensure adequate availability through proper infant nutrition. Although the free choline moiety is adequately provided by infant formulas and bovine milk, reevaluation of the concentrations of other choline esters, in particular glycerophosphocholine and phosphocholine, may be warranted.

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically. Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute. The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic-spectrophotometric procedure. An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.

Background: It was recently suggested that malignant hyperthermia-susceptible (MHS) patients could have an elevated peak of phosphodiesters in leg muscles using in vivo phosphorus magnetic resonance spectroscopy. In the current study, analysis of the phosphodiesters of muscle extracts of MHS and malignant hyperthermia-negative patients was performed using in vitro phosphorus magnetic resonance spectroscopy to chemically identify and to compare the muscle concentrations of water-soluble compounds between the two groups with respect to the muscle fiber type composition., Methods: Perchloric acid extracts of the vastus medialis muscle of seven MHS patients and ten malignant hyperthermia-negative patients on the basis of the European malignant hyperthermia contracture test were subjected to in vitro phosphorus magnetic resonance spectroscopy carried out at 9.4 T. In addition, chemical identification of the phosphodiester region and histologic examination of the muscle specimens were performed., Results: The peak in the phosphodiester region was assigned to glycerophosphorylcholine. Muscle perchloric acid extracts of MHS patients had a significantly (P < 0.05) higher glycerophosphorylcholine to the sum of phosphocreatine and inorganic phosphate (glycerophosphorylcholine/ [phosphocreatine +inorganic phosphate]) value than those of malignant hyperthermia-negative patients. Neither a difference in the fiber type composition between the two groups nor any specific myopathy were found., Conclusions: In the absence of histologic differences between muscle specimens of MHS and malignant hyperthermia-negative patients, these results could suggest that glycerophosphorylcholine could be a marker of an impairment in the phospholipid metabolism in the skeletal muscle of MHS patients.