Citrate Peak Research Articles

GABA shunt pathway is stimulated in response to early defoliation-induced carbohydrate limitation in Mandarin fruits

The regulation of sugar and organic acid metabolism during fruit development has a major effect on high-quality fruit production. The reduction of leaf area is a common feature in plant growth, induced by abiotic and biotic stresses and disturbing source/sink ratio, thus impacting fruit quality. Here, we induced carbohydrate limitation by partial leaf defoliation at the beginning of the second stage of mandarin development (before the citrate peak). Resulting changes were monitored in the short-term (48 h and 1 week) and long-term (7 weeks) after the defoliation. Short-term response to early defoliation implied metabolic settings to re-feed TCA for sustaining respiration rate. These features involved (i) vacuolar sucrose degradation (high acid invertase activity and mRNA expression level) and enhanced glycolytic flux (high ATP-phosphofructokinase activity), (ii) malic and citric acid utilization (increased phosphoenolpyruvate kinase and NADP-Isocitrate dehydrogenase) associated with vacuolar citric acid release (high mRNA expression of the transporter CsCit1) and (iii) stimulation of GABA shunt pathway (low GABA content and increased mRNA expression of succinate semialdehyde dehydrogenase). A steady-state proline level was found in ED fruits although an increase in P5CS mRNA expression level. These results contribute to a better knowledge of the molecular basis of the relationship between defoliation and sugar and organic acid metabolism in mandarin fruit.

Role of Multiparametric Magnetic Resonance Imaging in the Evaluation of Prostatic Pathologies on 3 Tesla Machine

Background and objectives Prostate diseases with an overall incidence of 9% are the leading cause affecting millions of males worldwide, with prostate cancer being the primary cause of death in males owing to benign prostatic hypertrophy being the primary cause of morbidity in the older age group. In light of this, we aimed to evaluate the utility of Magnetic Resonance Imaging in evaluating and characterization prostatic pathologies and grading prostatic cancer using PIRADS v2. Methods This was a study carried out on 50 patients with the age group ranging from 50 to 90 years with clinical suspicion or ultrasonographically demonstrable prostatic pathology and correlation of multiparametric MRI findings on various sequences with clinical features and histopathological findings and classification into groups of benign and malignant prostatic lesions and according to the PIRADS v2 scoring. Results Out of our study group, 4 cases were diagnosed with prostatitis and prostatic abscess, and 46 cases were categorized into PI-RADS, out of which most cases were classified into PI-RADS category V, followed by PI-RADS II and III. These were then evaluated with MR spectroscopy, of which 30 cases showed choline peak, and 16 cases showed citrate peak. The sensitivity and specificity for multiparametric MRI sequences combination detect the carcinoma of the prostate was calculated, and the Positive Predictive Value (PPV) & Negative Predictive Value (NPV) was found as 76% and 92%, respectively. Conclusion Based on our findings, we found out that the multiparametric MRI approach gives a wealth of practical information that has dramatically enhanced the detection and characterization of prostatic lesions. We also found out that morphological and functional sequencing improves the sensitivity and specificity of the lesions' identification and characterization.

(1)H NMR-based metabolite profiling workflow to reduce inter-sample chemical shift variations in urine samples for improved biomarker discovery.

Metabolite profiling of urine has seen much advancement in recent years, and its analysis by nuclear magnetic resonance (NMR) spectroscopy has become well established. However, the highly variable nature of human urine still requires improved protocols despite some standardization. In particular, diseases such as kidney disease can have a profound effect on the composition of urine and generate a highly diverse sample set for clinical studies. Large variations in pH and the cationic concentration of urine play an important role in creating positional noise within datasets generated from NMR. We demonstrate positional noise to be a confounding variable for multivariate statistical tools such as statistical total correlation spectroscopy (STOCSY), thereby hindering the process of biomarker discovery. We present a two-dimensional buffering system using potassium fluoride (KF) and phosphate buffer to reduce positional noise in metabolomic data generated from urine samples with various levels of proteinuria. KF reduces positional noise in citrate peaks, by decreasing the mean relative standard deviation (RSD) from 0.17 to 0.09. By reducing positional noise with KF, STOCSY analysis of citrate peaks saw significant improvement. We further aligned spectral data using a recursive segment-wise peak alignment (RSPA) method, which leads to further improvement of the positional noise (RSD = 0.06). These results were validated using diverse selection of metabolites which lead to an overall improvement in positional noise using the suggested protocol. In summary, we provide an improved workflow for urine metabolite biomarker discovery to achieve higher data quality for better pathophysiological understanding of human diseases. Graphical abstract Citrate peaks in the range 2.75-2.5ppm from datasets with different sample preparation protocols and with/without in silico alignment. A Citrate peaks with standard phosphate buffering and without in silico alignment. B citrate peaks with standard phosphate buffering and with in silico alignment. C citrate peak with additional potassium fluoride and standard phosphate buffering without in silico alignment. D citrate peaks with additional potassium fluoride and standard phosphate buffering with in silico alignment. Below the respective spectrum are displayed the percent relative standard deviation (RSD) of the respective citrate peaks. This is a measure of the positional noise of peaks within a (1)H NMR analysis. It can be seen that D performs the best in reducing positional noise of citrate peaks. E-H STOCSY analysis of correlating spectral features with the driver peak at 2.675ppm (see red arrow) to identify structural correlations. As a, b, c, and d are known to be structurally correlated, STOCSY analysis should reveal r (2) = 1 if data is perfectly aligned and can therefore be used as a measure of peak alignment. E Strong positional noise does not allow identifying the c and d peaks of the AB system to be correlated. F, G Neither in silico alignment or KF addition alone can completely improve the alignment and therefore increase the correlations. H Highly improved alignment by combining both KF addition and in silico alignment reduces positional noise and elucidates all four citrate peaks to be strongly correlated.

Endorectal coil MRI and MR-spectroscopic imaging in patients with elevated serum prostate specific antigen with negative trus transrectal ultrasound guided biopsy

Background:The aim of this study was to see the efficacy of endorectal coil MRI and MR spectroscopic imaging in patients with elevated serum PSA and negative transrectal ultrasonography (TRUS)-guided biopsy.Materials and Methods:This study was conducted on 87 patients presented with: • Elevated prostatic specific antigen levels >5 ng/ml • Symptoms and signs of prostatic carcinoma • Patients with negative TRUS-guided biopsy • Suspicious lesion on TRU. All the patients were subjected to TRUS and followed by TRUS-guided biopsy of the lesion identified on endorectal coil MRI and MR-Spectroscopy. TRUS-guided biopsy of prostate was done with a Siemens Sonoline Adana Scanner. The scanning was performed by mechanical probe 5-7.5 MHz.Results:Out of 87 patients, 43 (49.4%) had hypointense lesion, 11 (12.6%) had hyperintense lesion. Out of 87 patients, MR-spectroscopy showed peak choline-creatine in 74 patients. Normal citrate peak was seen in 13 patients. Patients who had choline-creatine peak, among them 28 (37.8%) had peak in left peripheral zone, 23 (31.1%) had peak in the right peripheral zone, 2 (2.7%) had peak in the central zone, 17 had (23%) peak bilaterally. Four patients (5.4%) had peaks in right and central zones. The difference was statistically significant (P < 0.001).Conclusion:Prostatic biopsy directed with endorectal coil MRI and MR-spectroscopic imaging findings in patients with elevated serum PSA and prior negative biopsy, improves the early diagnosis of prostatic carcinoma and accurate localization of prostate cancer within the gland.

Prostate 3T-MR spectroscopic imaging without an endorectal surface coil using the MLEV-PRESS sequence

The aim of this study was to determine to what extent we could identify the metabolism product peak using 3-T proton magnetic resonance spectroscopic imaging (MRSI) of the prostate gland in healthy volunteers by combining an external array coil with Malcolm Levitt composite-pulse decoupling sequence (MLEV)-point-resolved spatially localized spectroscopy (PRESS). MRSI data were obtained from the entire prostate gland in six healthy volunteers. The heights of the choline, citrate peaks and the standard deviation (SD) of the noise in each voxel were calculated. When the choline and/or citrate peak in a voxel exceeded 3 SD, the peak was clearly identified. The clear citrate peak rate in the peripheral zone (PZ) and the central gland (CG) were 78.8 and 70.3 %, respectively. The clear choline peak rate in the PZ and the CG were 55.4 and 44.9 %, respectively. In addition, the clear peak rates for both citrate and choline in the PZ and the CG were 51.8 and 38.6 %. Therefore, in the entire prostate gland, 75.2 % had a clear citrate peak, 51.1 % had a clear choline peak, and 46.3 % had both citrate and choline peaks. The citrate peak was clearly detected in 75.2 % of the voxels by this technique.

Locally Advanced Prostate Cancer: Three-Dimensional Magnetic Resonance Spectroscopy to Monitor Prostate Response to Therapy

To correlate results of three-dimensional magnetic resonance spectroscopic imaging (MRSI) with prostate-specific antigen (PSA) levels and time since external beam irradiation (EBRT) in patients treated with long-term hormone therapy (HT) and EBRT for locally advanced disease to verify successful treatment by documenting the achievement of metabolic atrophy (MA). Between 2006 and 2008, 109 patients were consecutively enrolled. MA was assessed by choline and citrate peak area-to-noise-ratio <5:1. Cancerous metabolism (CM) was defined by choline-to-creatine ratio >1.5:1 or choline signal-to-noise-ratio >5:1. To test the strength of association between MRSI results and the time elapsed since EBRT (TEFRT), PSA levels, Gleason score (GS), and stage, logistic regression (LR) was performed. p value <0.05 was statistically significant. The patients' outcomes were verified in2011. MRSI documented MA in 84 of 109 and CM in 25 of 109 cases. LR showed that age, GS, stage, and initial and recent PSA had no significant impact on MRSI results which were significantly related to PSA values at the time of MRSI and to TEFRT. Patients were divided into three groups according to TEFRT: <1 year, 1-2 years, and >2 years. MA was detected in 54.1% of patients of group 1, 88.9% of group 2, and in 94.5% of group 3 (100% when PSA nadir was reached). CM was detected in 50% of patients with reached PSA nadir in group 1. Local relapse was found in 3 patients previously showing CM at long TEFRT. MA detection, indicative of successful treatment because growth of normal or abnormal cells cannot occur without metabolism, increases with decreasing PSA levels and increasing time on HT after EBRT. This supports long-term HT in advanced prostate cancer. Larger study series are needed to assess whether MRSI could predict local relapse by detecting CM at long TEFRT.

The influence of EDTA and citrate anticoagulant addition to human plasma on information recovery from NMR-based metabolic profiling studies

The widely-used blood anticoagulants citrate and EDTA give rise to prominent peaks in (1)H NMR spectra of plasma samples collected in epidemiological and clinical studies, and these cause varying levels of interference in recovering biochemical information on endogenous metabolites. To investigate both the potential metabolic information loss caused by these substances and any possible inter-molecular interactions between the anticoagulants and endogenous components, the (1)H NMR spectra of 40 split human plasma samples collected from 20 individuals into either citrate or EDTA have been analysed. Endogenous metabolite peaks were selectively obscured by large citrate peaks or those from free EDTA and its calcium and magnesium complexes. It is shown that the endogenous metabolites that give rise to peaks obscured by those from EDTA or citrate almost invariably also have other resonances that allow their identification and potential quantitation. Also, metabolic information recovery could be maximised by use of spectral editing techniques such as spin-echo, diffusion-editing and J-resolved experiments. The NMR spectral effects of any interactions between the added citrate or EDTA and endogenous components were found to be negligible. Finally, identification of split samples was feasible using simple multivariate statistical approaches such as principal components analysis. Thus even when legacy epidemiological plasma samples have been collected using the NMR-inappropriate citrate or EDTA anticoagulants, useful biochemical information can still be recovered effectively.

SU‐FF‐J‐177: Effect of Gold Marker Seed On MR Spectroscopy of the Prostate

Purpose: Prostate magnetic resonance stereoscopic imaging (MRSI) is an emerging technology that may enhance targeting in radiotherapy. Gold seed fiducial markers are commonly used in prostate radiotherapy to perform daily prostate localization. If MRSI is to be used in targeting prostate cancer, the impact of gold seeds on MRSI must be understood. The purpose of this study is to characterize the local effects of gold seeds on the chemical shift spectra of MRSI. Method and Materials: Two different phantoms are used in this study. One is filled with water and agar containing Choline. A Gold Marker is seed is put in the agar. In the second phantom a cylindrical cavity is filled with liquid Choline and Citrate in water. A gold Marker seed is mounted in the middle of the fluid. Single voxel (SV) and three‐dimensional MR Spectra of the chemical shift (CSI) are taken. Chemical Shift spectra are compared in the presence and absence of gold seed. Spectra close to and near the seed are studied. Results: In general, Choline and Citrate peaks are broadened by the single voxel containing the seed. Local effects on adjacent voxels are seen, mostly by way of a altered baseline drift. This artifact dissipates with distance and is most prominent when the gold seed is aligned perpendicular to the magnetic field. A precise description of the gold seed artifact on prostate MRSI chemical shift spectra will be reported. Conclusion: Gold seeds can cause local artifacts in prostate MRSI. Characterization of this artifact will permit more accurate interpretation of the MRSI spectra.

Combined MRI and MRS in Prostate Cancer: Improvement of Spectral Quality by Susceptibility Matching

Local magnetic field inhomogeneity caused by susceptibility artifacts due to air in the endorectal coil substantially degrades the quality of 3D MR spectroscopic imaging (3D-MRSI). Perflubron (PFB) has magnetic susceptibility similar to that of human tissue. We prospectively assessed the effect of susceptibility matching using PFB on in vivo prostate (1)H-3D-MRSI. Ninety-one consecutive patients referred for 3D-MRSI were examined using air and PFB as the filling agent for endorectal coils at 1.5T with an identically placed PRESS box and sat bands. Solely auto-shim without additional manual shimming was used. The full width at half maximum (FWHM) of the water peak was statistically compared with a paired t-test. The spectral quality was visually evaluated for the definition of metabolite peaks and for the citrate peak split (duplet). The MR image quality was rated on a five-point scale. FWHM was significantly less (p < 0.001) using PFB (mean 9.0 +/- 3.3, range 3 - 20) than air (mean 14.9 +/- 4.2, range 6 - 26) in 85/91 patients (93%). The spectral quality markedly improved using PFB and frequently the duplet of the citrate peak was able to be identified. Image quality ratings were similar (mean rating PFB 4.2, air 4.3 points). Omitting manual shimming led to a time savings of 4 min. per study. 3D-MRSI using PFB for susceptibility matching benefits from significantly better local field homogeneity, thus providing improved spectra quality. Combined with a substantial time savings in data acquisition, this may increase the clinical utilization of 3D-MRSI in patients with prostate cancer.

Controlling the Surface Morphology of ZnO Films Grown on Glass Substrates by Hydrothermal Method at 90 °C: Effects of Na-Citrate on the Shape of ZnO Nano-Crystallites

The effects of Na-citrate on the morphology change of ZnO films, grown on ZnO buffered glass substrates by hydrothermal synthesis at 90 degrees C and at pH 10.9, have been investigated. Dense and smooth ZnO film consists of ZnO nano-rods that have flat ends was grown with Na citrate. However, very rough ZnO film consists of ZnO nano-rods that have sharp ends were grown without Na citrate. X-ray diffraction analysis shows that all the ZnO films were grown with strong c-axis out-of-plane orientation. Optical transmission spectroscopy of the ZnO films grown with and without Na-citrate shows band gap energy values of 3.36 eV and 3.28 eV, respectively. Photoluminescence results showed strong defect related emission peak centered near 545 nm in the ZnO film grown without Na citrate and strong band-edge emission peak centered near 378 nm in the ZnO film grown with Na citrate.

Performance Characteristics of an Ion Chromatographic Method for the Quantitation of Citrate and Phosphate in Pharmaceutical Solutions

The performance of an ion chromatographic method for measuring citrate and phosphate in pharmaceutical solutions is evaluated. Performance characteristics examined include accuracy, precision, specificity, response linearity, robustness, and the ability to meet system suitability criteria. In general, the method is found to be robust within reasonable deviations from its specified operating conditions. Analytical accuracy is typically 100 +/- 3%, and short-term precision is not more than 1.5% relative standard deviation. The instrument response is linear over a range of 50% to 150% of the standard preparation target concentrations (12 mg/L for phosphate and 20 mg/L for citrate), and the results obtained using a single-point standard versus a calibration curve are essentially equivalent. A small analytical bias is observed and ascribed to the relative purity of the differing salts, used as raw materials in tested finished products and as reference standards in the analytical method. The assay is specific in that no phosphate or citrate peaks are observed in a variety of method-related solutions and matrix blanks (with and without autoclaving). The assay with manual preparation of the eluents is sensitive to the composition of the eluent in the sense that the eluent must be effectively degassed and protected from CO(2) ingress during use. In order for the assay to perform effectively, extensive system equilibration and conditioning is required. However, a properly conditioned and equilibrated system can be used to test a number of samples via chromatographic runs that include many (> 50) injections.

Proton magnetic resonance spectroscopy with a body coil in the diagnosis of carcinoma prostate.

We evaluate the feasibility of proton magnetic resonance spectroscopy ((1)H MRS) using a spine coil receiver (body coil) in the diagnosis of carcinoma prostate. Seventeen patients with biopsy-proven prostate carcinoma, five patients of benign prostatic hyperplasia (BPH) and five healthy young volunteers underwent (1)H MRS investigation. MRS was performed at 1.5 Tesla using a spine receiver coil for signal reception. In vivo citrate levels are reported as a ratio of citrate peak area to the sum of the areas of choline and creatine peak. MRS spectrum with good sensitivity and signal to noise (S/N) ratio was obtained in all 27 subjects. The citrate to creatine plus choline ratio was 0.31+/-0.25 in patients with cancer, 1.43+/-0.58 in BPH and 2.16+/-0.56 in controls. The difference in ratios between cancer and BPH and cancer and control was statistically significant ( p<0.01). Within the cancer group, there was a statistically significant decline in levels with higher-grade malignancy ( p<0.05). There were no complications of the procedure. There is a statistically significant decline in the ratio of citrate to choline plus creatine in the regions of cancer prostate when compared with BPH or normal control. The study demonstrates that MRS data can be reliably acquired using a spine coil receiver. MRS may also play a role in differentiating well-differentiated tumors from the anaplastic variety.

Citrate pharmacokinetics and metabolism in cirrhotic and noncirrhotic critically ill patients.

To investigate pharmacokinetics and metabolism of sodium citrate in critically ill patients. To determine the risk of citrate accumulation in the setting of liver dysfunction (cirrhosis, hepatorenal syndrome). Prospective cohort study. Intensive Care Unit, Department of Medicine IV, University Hospital Vienna. Consecutive critically ill cirrhotic (n = 16) and noncirrhotic patients (n = 16). Infusion of sodium citrate (0.5 mmol.kg-1.hr-1) and calcium chloride (0.17 mmol.kg-1.hr-1) for 2 hrs. Analysis of serial arterial blood samples. Total body clearance of citrate was normal in noncirrhotic critically ill patients but significantly reduced in cirrhotic patients (710 vs. 340 mL/min, p =.008). Citrate peak concentrations and concentration over time were increased by 65% and 114% in cirrhotic patients (p <.001), respectively; volumes of distribution were similar. Net metabolic changes were quantitatively similar, with pH and plasma bicarbonate concentrations increasing more slowly in cirrhotic patients. No citrate-related side effects were noted. Citrate clearance could not be predicted by standard liver function tests and was not appreciably influenced by renal function and Acute Physiology and Chronic Health Evaluation II scores. This first systematic study on citrate pharmacokinetics and metabolism in critically ill patients confirms a major role of hepatic citrate metabolism by demonstrating reduced citrate clearance in cirrhotic patients. Pharmacokinetic data could provide a basis for the clinical use of citrate anticoagulation in critically ill patients. Provided dose adaptation and monitoring of ionized calcium, citrate anticoagulation seems feasible even in patients with decompensated cirrhosis. Metabolic consequences of citrate infusion were not different between groups in this study but may be more pronounced in prolonged infusion.

Gastrointestinal absorption of aluminium and citrate in man

Aluminium (Al) is an abundant terrestrial element, but toxic to tissues, including brain. The body is largely protected because systemic Al absorption is very low and in normal individuals almost all absorbed Al is excreted from the body. However gastrointestinal (Gl) absorption is enhanced by organic acids, including citrate. Aluminium and citrate Gl absorption was measured in three healthy males, aged 40–46. After overnight fast, subjects drank a 100 ml fruit drink containing 280 mg Al and 3.2 g citrate (104 and 167 mM, respectively). Al was measured in timed blood and urine samples by GFAAS and serum citrate by enzymatic assay. Blood Al peaked by an increase of 13 ± 2.1 μg/l after 87 ± 19 min then fell slowly over 24 h. Plasma citrate peaked after 32 min, returning to baseline by 90 min. Al was excreted at a constant rate for the first 24 h, 0.4% of the dose being excreted in urine by this time. It is unlikely that Al is absorbed as Al citrate because the blood citrate peak preceded the Al peak by 45–60 min.

Capillary zone electrophoresis with UV‐scanning detection for the analysis of metal species and their organic binding partners in biological systems

AbstractA capillary zone electrophoretic method with UV‐scanning detection for the separation and identification of both free ligands and metal species is presented. The electrophoretic behavior of naturally occurring binding partners such as organic acids and amino acids was studied and compared with their metal‐complexes. Copper(II) complexes of citrate and amino acids showed decreased electrophoretic mobilities and altered UV spectra. The optimized method was validated on ultrafiltered cow's milk and human milk samples. In cow's milk six low molecular weight substances, including citrate, orotate, and hippurate, could be separated. Metal supplementation with Cu(II), Ni(II), or Zn(II) decreased not only the citrate peak but also the orotate signal and in the case of copper(II) supplementation a new signal for the copper(II)‐citrate complex appeared. In human milk samples various amino acids such as glutamate, phenylalanine, tyrosine, and tryptophan could be identified besides citrate. The electrophoretic mobilites and peak areas of the amino acids were also influenced by the metal supplementation.

Quantification of citrate concentration in the prostate by proton magnetic resonance spectroscopy: zonal and age-related differences.

A commercial phased-array multicoil was used to acquire water-suppressed localized proton spectra of the two major anatomical regions of the prostate. The signal-to-noise ratio and spectral resolution allowed identification of peaks from choline and creatine, as well as a major peak from citrate. Quantification of the citrate peak using experimentally determined relaxation parameters with tissue water as an internal concentration reference revealed a marked variability between different volunteers. Nevertheless, in each case, the citrate concentration was up to fourfold greater in the peripheral zone than in the central gland. Furthermore, the difference in citrate concentration between these two regions was positively correlated with the subjects age. The results indicate a consistent difference in cellular function between the major anatomical regions within the prostate and may have important consequences for the application of magnetic resonance spectroscopy to the diagnosis of prostatic pathology.

Quantitative proton MRS assessment of citrate heterogeneity in normal prostate

Water-suppressed proton spectra of the two major anatomical regions of the normal prostate were acquired with a commercial phased-array multicoil. The spectra demonstrated excellent signal-to-noise ratio and spectral resolution allowing identification of peaks from choline, creatine, and amino acids, as well as a major peak from citrate. Quantification of the citrate peak using tissue water as an internal concentration reference revealed a marked variability among different volunteers. In each case, citrate was consistently twofold to threefold greater in the peripheral zone than in the central gland. The results demonstrate heterogeneity of citrate within the prostate and suggest significant differences in the cytology and hormonal control of citrate synthesis among individuals.

Imaging and localized spectroscopy of 13C by polarization transfer

The biological applications of 13C magnetic resonance spectroscopy are well known ( 1-5). Recently, there was evidence (6-9) suggesting that the citrate peak in the 13C spectrum may be useful as a marker for human prostate cancer. It was found in human prostate that the citrate concentration is small in the normal gland, negligible in ma lignant tumors (8)) and increased markedly in tissues with benign prostatic hypertrophy (BPH) (8, 9). Wh ile this evidence suggests citrate as possibly a specific marker for ma lignant prostatic tumor and BPH, it also underscores the problem in applying NMR spectroscopic techniques without localization under in vivo conditions where prostatic ma lignancy is often, if not always, accompanied by BPH. There are two possible solutions to the above problem. One is through mu ltidimensional chemical-shift imaging ( 10, I I ) and the other, localized spectroscopy. For carbon, either one of these two approaches presents many challenging problems. F irst, one must deal with the problem of low sensitivity and long T1. Second, because carbon has a low y, one needs four times as much gradient strength in imaging or localization of carbon as that of hydrogen. Third, since carbon has large chemical shifts, it is often difficult to separate the spatial from the spectral information. By using polarization transfer techniques, such as INEPT ( 12,13) or DEPT ( 14,15), the problems associated with low y nuclei, such as carbon, are converted into those of protons which are much easier to solve. Recently this idea was exploited by Norris et al. ( 16)) who applied a one-dimensional ISIS sequence ( 17) on the protons for the localization of carbon. In this Communication, we will demonstrate an alternative technique of carbon selection based on the idea ment ioned above and the conventional p lane excitation method commonly used in proton imaging. In this scheme, plane selection of carbon is obtained by selective proton enhancement. We will also demonstrate that one can gain a similar type of enhancement in carbon imaging by applying phase encoding on the proton, instead of the carbon, polarization. We shall call this an indirect spinwarp method. F igure 1 shows the method of localization of carbon by selective enhancement through excitation of hydrogen. The RF section of the pulse sequence in F ig. 1 is a DEPT sequence with a shaped proton 7r/2 pulse. Since the phase-cycl ing scheme of the DEPT sequence is so designed that only the proton-enhanced nuclei are observed

Determination of Urinary Citrate by Ion Chromatography

Ion chromatography was used to determine urinary citrate concentration. The technique is simple, requiring a 100-fold dilution of urine and 35 minutes at most for each analysis. The technique does, however, have the disadvantage that it requires a rather expensive automated system. The minimum detectable limit for citrate was 0.5 μg./ml. in a standard solution, and the regression line for the standard curve from 0.5 to 50μg./ml. citrate had a significant correlation coefficient (lnY = 1.048 × lnX-2.755, r = 0.997). The intra-run coefficient of variation was 0.9 per cent. The overall intra-run and inter-run coefficients of variation, including the sampling and dilution of urine, were 4.8 and 8.3 per cent respectively. There is a possibility that another ion is close enough to the citrate peak to interfere; however, this problem can be solved by the treatment of the sample with citrate lyase. When the present method was compared with the conventional enzyme method, a significant correlation between the results was obtained in both human and rat urine. The 24-hour urinary citrate excretion in eight normal human males was 73.8 to 378.4mg./day (mean, 174.7mg./day), while that in male Wistar-strain rats (approximately 150gm.) was 1.5 to 18.6mg./day (mean, 9.5mg./day), as measured in more than 50 consecutive rat urine samples.

Simultaneous 13C and 31P NMR studies of perfused rat liver. Effects of insulin and glucagon and a 13C NMR assay of free Mg2+.

Metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+ in the presence and absence of 7 nM insulin has been followed at 35 degrees C by alternate scan 13C and 31P NMR at 90.5 and 145.8 MHz, respectively, in isolated perfused liver from 16-h fasted rats. With this technique, 31P and 13C NMR spectra are recorded simultaneously so that both phosphate metabolites and 13C-labeled metabolites could be followed, noninvasively, in perfused liver to give a comprehensive view of the response to a variety of stimuli. 13C-labeled glycogen increased synchronously, at a rate of 17 mumol of glucose units/g of liver/h, with the synthesis of 13C-labeled glucose, which also proceeded at a rate of 17 mumol/g of liver/h; glycogenesis was essentially a gluconeogenic process under these conditions and was not affected by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of Kd for the citrate-Mg complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM in perfused rat liver. After subsequent administration of glucagon, a rapid decrease in glycogen and citrate was seen by 13C NMR and a 44% increase in glycero-3-phosphocholine was seen by 31P NMR; increase in glycero-3-phosphocholine is consistent with stimulation of liver phospholipase activity by glucagon. The co-administration of two different 13C-labeled substrates introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. 13C enrichments at specific carbons of citrate, glutamate, glutamine, beta-hydroxybutyrate, and glucose and the distribution of intensity within the multiplets of specific carbons were measured in spectra of perfusates and extracts of the freeze-clamped livers. Within the context of a first order model for fluxes into the Krebs cycle and into glucose, analytical expressions were written that describe the intensity distributions within the several multiplets. In this way, a set of simultaneous equations was generated and solved in general form; when the measured intensity ratios are substituted into these expressions, relative fluxes under the conditions of the experiment can be estimated. Because a redundancy of information is available, checks on self-consistency are built into the estimated fluxes.