TBDMS Derivatives Research Articles

Chemico-pharmacological evaluation of the methanolic leaf extract of Catharanthus ovalis: GC-MS/MS, in vivo, in vitro, and in silico approaches.

Introduction: Natural plant-based medicines have gained popularity recently as a major source of inventive, risk-free, and more potent secondary bioactive compounds with medicinal potential. Catharanthus ovalis is a perennial shrub containing various indole alkaloids cultivated extensively for local medical purposes. Methods: This research is conducted to identify the phytocompounds present in the leaves of C. ovalis and its central and peripheral analgesic, thrombolytic, and membrane-stabilizing activities through tail immersion, acetic acid-induced writhing, human blood clot lysis, and erythrocyte lysis by heat and hypotonic solution methods, respectively. Results and discussion: A total of 39 compounds were identified using GC-MS/MS techniques, including hexadecanoic acid, methyl ester (56.749%), methyl stearate (29.782%), carvacrol and its TBDMS derivative (12.586%), and 9-octadecenoic acid, methyl ester, (E)-] (9.297%) presented in high quantity. The highest tail immersion latency was observed for the 600mg/kg extract of C. ovalis crude extract. Both 400 and 600mg/kg doses of C. ovalis crude extract exhibited prominent peripheral analgesic activity. The maximum thrombolytic effect was observed by DCM soluble fraction extract by inhibiting 54.87% of the clot. However, the aqueous-soluble fraction of this extract manifested an excellent membrane-stabilizing effect by showing 73.98% and 87.51% hemolysis against heat- and hypotonic-induced hemolysis, respectively. Some of the compounds were identified as active agents against different receptors related to these diseases, which supported the findings of in vitro and in vivo tests. Conclusion: Further investigation needs to be conducted to specify and identify the exact mechanism of action of these compounds.

GC-MS Compound Identification in Phaseolus vulgaris - A Low-Cost Cataract Prevention Food

Phaseolus vulgaris is one of the most widely used traditional remedies for diabetes mellitus. Over a thousand medicinal plants have been found to contain anti-diabetic chemicals, the bulk of which have long been used in traditional medicine. The pharmacological significance of eating Phaseolus vulgaris in diabetics has been intensively explored in order to identify the several bioactive components responsible for their diabetes-controlling benefits. Researchers have previously linked the eating of P. vulgaris as a possible protective factor against cataract surgery based on their findings on the evaluation of beans and other vegetables. This study was driven by the need to find more bioactive chemicals that are active in the management of diabetes and the prevention of cataract, which is one of the disease's consequences. In Ado-Ekiti, Nigeria, Phaseolus vulgaris is a leguminous crop that is widely consumed. Phaseolus vulgaris was purchased from two different sources, pulverized with a Sumeet CM/L 2128945 grinder, and extracted with methanol and ethanol. The extracts were concentrated. The bioactive components in the extracts were identified using GC-MS. The ethanolic extract includes twelve bioactive compounds, while the methanolic extract contains ten. The ethanol extract contains neophytadiene, β-amyrin, and squalene, among other chemicals. Neophytadiene, 1,2-benzisothiazol-3-amine, TBDMS derivative, and 1-Methyl-3-phenylindole were found in methanol extract. Both extracts contain bioactive chemicals that have been shown to have anti-diabetic and cataract-prevention properties in humans. Finally, the discovery of these various beneficial components lends credence to scientific proof that Phaseolus vulgaris can be consumed by diabetics.

Method for the Improvement of Enrichment Estimation in Stable Isotope Metabolic Studies

BackgroundStable isotope methods are currently the most accurate in vivo approach to estimate protein kinetics. Tracers are molecules where atoms on their structure have been exchanged with heavier isotopes. The determination of substrate kinetics is possible through the administration of a tracer molecule and subsequent measurement of enrichment (ratio of tracer‐to‐tracee, TTR) over time. The attachment of heavier atoms on molecules results in a shift in the tracer's mass distribution therefore, mass spectrometry readings should be processed to obtain the true TTR. Usually, the correction is done by applying the “skew” correction factor. While improvement is achieved, differences remain between theoretical estimations and actual experimental results. We hypothesize that accounting for tracer's isotopic purity would further improve TTR estimation.MethodsThe L‐[ring‐13C6] Phenylalanine (Cambridge Isotope Laboratories, Andover, MA, USA) with nominal isotopic purity of 99% was used. The TBDMS derivative was prepared and signal intensities at mass spectra 340, 341, 342, 343 (M4, M5, M6, M7) (corresponding to the fragment C17H30N1Si2) were measured using GC‐MS (model 5973, Hewlett‐Packard Co., Palo Alto, CA). Using the binomial and multinomial distributions the most likely value of isotopic purity of the tracer was estimated. Subsequently, three mixtures of unlabeled phenylalanine with varying amounts of tracer were prepared yielding samples with TTR of 0.5%, 4% and 20% respectively. The samples were processed as described above and intensities at mass spectra 336 (M0), 341 (M5) and 342 (M6) were measured. Having established the tracer's isotopic purity, intensities at M0, M5 and M6 were modeled as linear combination of tracer and tracee masses using least squares method and TTR was estimated as the ratio of the two regression coefficients. TTR was also estimated with the traditional method by applying the “skew” correction factor to the signal intensities ratio M6/M0.ResultsThe tracer's isotopic purity was estimated at 99.1%, corresponding to 71% and 4.7% of relative mass at mass numbers 342 (M6) and 341 (M5) respectively. The estimated TTR of the samples with known TTR of 0.5%, 4% and 20% using the traditional method were 0.46%, 3.75% and 19.1% (error of 8%, 6.25% and 4.5%) respectively. With the new method estimated TTR were 0.52%, 3.97%, 20.2% (errors of 4%, 0.7%, 1%) respectively.ConclusionsOur preliminary data suggest mild improvement in TTR estimation. We expect the new method to significantly improve TTR estimates in samples containing multiple tracers with overlapping spectra and more accurate determination of muscle protein kinetics calculated through complicated formulas and rely on exponential decay modeling.Support or Funding InformationThis work was supported by grants from NIH (P50 GM060338, R01 GM056687, T32 GM008256, R01 HD049471, K01 HL070451), NIDILRR (90DP00430100), Shriners of North America (SHC 84080, SHC 84090)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Advances in the alkylsilyl derivatization of glyphosate and aminomethylphosphonic acid: A critical comeback to the N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide reagent

A novel, full tert.-butyldimethylsilylation approach - derivatizing N-(phosphonomethyl) glycine (glyphosate, GLYP) and its principal metabolite aminomethylphosphonic acid (AMPA) - was noted. The full labeling with the N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) reagent was triggered, extending the mass acquisition up to the m/z 1000 values. Under this condition, the 4.TBDMS species - for GLYP and AMPA equally - were recognized and determined, at first. Following their mass fragmentation properties by gas chromatography mass spectrometry (GC/MS) the not yet described molecular ions (GLYP.4TBDMS: m/z 625, and AMPA.4TBDMS: m/z 567), along with their characteristic decomposition products were confirmed.The complementary impact of solvents (acetonitrile, ACN, pyridine, PYR) was also studied. PYR, for the time being not yet applied with MTBSTFA proved to be also suitable in the tert.-butyldimethylsilyation process. Along with ACN both support the 4.TBDMS derivative formation: ensuring an improved selectivity and sensitivity of the protocol, for both species examined.As to the analytical performance characteristics, under optimum conditions, LOQ values proved to be close to each other: in MTBSTFA/ACN medium for GLYP 1.18ng/μL for AMPA 0.036ng/μL was confirmed. In MTBSTFA/PYR medium for GLYP 2.36ng/μL, for AMPA 0.036ng/μL values were determined. It is worth to note that AMPA.4TBDMS ions in the MTBSTFA/ACN medium show up above 0.14ng/μL while, in the MTBSTFA/PYR medium above 0.072ng/μL: meaning that PYR favors their formation. Repeatability of model measurements, characterized with the relative standard deviation percentages (RSD%), varied from 0.39 RSD% up to 5.9 RSD% with an average of 2.97 RSD%.Practical utility of the MTBSTFA/PYR method was confirmed by the analysis of the AMPA impurities in GLYP market samples, expressed as g/L AMPA: GlialkaR Star, 6.45g/L (5.1 RSD%); FozátR, 6.89g/L (4.34 RSD%); Medallon P, 7.15g/L(4.28 RSD%) and Total Spray, 0.038g/L (4.64 RSD%).

Effects of leucine and its metabolite β‐hydroxy‐β‐methylbutyrate on human skeletal muscle protein metabolism

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses–fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is ‘sensed’ have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-13C2]Leu and [2H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A–V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70%vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling ≤90 min vs. ≤30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; −57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Injection port silylation of γ-hydroxybutyrate and trans-hydroxycrotonic acid: Conditions optimisation and characterisation of the di-tert-butyldimethylsilyl derivatives by GC-MS

Silylation is usually carried out on γ-hydroxybutyrate (GHB) for its analysis by Gas Chromatography/Mass Spectrometry (GCMS) and requires potentially long incubation times before injection during which the derivatisation reagent and derivatives (such as trimethyl-silyl compounds) can hydrolyse. Moreover, alternative internal standards (IS) are often useful depending on sample matrices, extraction/purification procedures, commercial availability and price. This study evaluated the possibility of silylating GHB with an injection port derivatisation procedure using N-methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (TBCS) as the derivatisation reagent, producing di-tert-butyldimethyl-silyl derivatives as a novel means of analyzing GHB. In parallel, trans-hydroxycrotonic acid (t-HCA) was investigated as a potential IS for GHB quantification. Analyses were carried out with a temperature programmable injector and the GHB(t-BDMS)(2) and t-HCA(t-BDMS)(2) derivatives were successfully produced, characterised and derivatisation conditions optimised. t-HCA behaved very similarly to GHB through the derivatisation processes and was used as the IS for the determination of urinary endogenous GHB concentrations in human subjects where the method showed a limit of detection of 0.049 μg mL(-1), a limit of quantification of 0.162 μg mL(-1), and a limit of confirmation of 1.33 μg mL(-1), suitable for toxicological GHB concentration determination.

ChemInform Abstract: Synthesis of Non-Enolizable α- and β-Peltatins.

The preparation of non-enolizable derivatives of both α- and β-peltatins via reaction of the lactone enolates of the corresponding TBDMS derivatives with CH3I and hexachloroethane is described. These derivatives are considerably less toxic than the precursor peltatins but show, at best, only marginal activity in in vivo tests against P 388 leukemia.

Derivatization of organophosphorus nerve agent degradation products for gas chromatography with ICPMS and TOF-MS detection

Separation and detection of seven V-type (venomous) and G-type (German) organophosphorus nerve agent degradation products by gas chromatography with inductively coupled plasma mass spectrometry (GC-ICPMS) is described. The nonvolatile alkyl phosphonic acid degradation products of interest included ethyl methylphosphonic acid (EMPA, VX acid), isopropyl methylphosphonic acid (IMPA, GB acid), ethyl hydrogen dimethylamidophosphate sodium salt (EDPA, GA acid), isobutyl hydrogen methylphosphonate (IBMPA, RVX acid), as well as pinacolyl methylphosphonic acid (PMPA), methylphosphonic acid (MPA), and cyclohexyl methylphosphonic acid (CMPA, GF acid). N-(tert-Butyldimethylsilyl)-N-methyltrifluroacetamide with 1% TBDMSCl was utilized to form the volatile TBDMS derivatives of the nerve agent degradation products for separation by GC. Exact mass confirmation of the formation of six of the TBDMS derivatives was obtained by GC-time of flight mass spectrometry (TOF-MS). The method developed here allowed for the separation and detection of all seven TBDMS derivatives as well as phosphate in less than ten minutes. Detection limits for the developed method were less than 5 pg with retention times and peak area precisions of less than 0.01 and 6%, respectively. This method was successfully applied to river water and soil matrices. To date this is the first work describing the analysis of chemical warfare agent (CWA) degradation products by GC-ICPMS.

Determination of ethylene oxide–hemoglobin adduct by silylation and gas chromatography–electron impact-mass spectrometry

A gas chromatography-electron impact ionization-mass spectrometric (GC-EI-MS) assay was developed for the determination of ethylene oxide-hemoglobin adduct (N-2-hydroxyethylvaline, HEVal). HEVal and deuterated HEVal (d(4)-HEVal) were synthesized for identification and quality control. Globin samples were separated from red blood cells (RBCs) by acidic isopropanol and extracted with ethyl acetate. HEVal adduct in globin was transformed to HEVal-pentafluorophenylthiohydantoin derivative by modified Edman-degradation method, which was extracted from globin with diethylether. d(4)-HEVal was used as an internal reference standard. The dried extract was derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)-NH(4)I (1000:4, w/w) containing 0.4 mg of dithioerthritol. The TBDMS derivative of HEVal had very good chromatographic property and offered sensitive response of GC-EI-MS. The recovery of HEVal was about 81.6% and the coefficient of variation was 5.0% at the concentration of 311 pmol/g. Low limit of detection (LOD) of the assay was 1.8 pmol/g in 0.1g hemoglobin. The experiments have demonstrated to detect background level of HEVal adduct in human blood. HEVal adduct in globin was detected between 12 and 6573 pmol/g.

Analysis of t-Butyldimethylsilyl Derivatives of Chlorophenols in the Atmosphere of Urban and Rural Areas in East of France

A method using GC-MS and derivatization with N-(t-butyldimethylsilyl)-N-ethyltrifluoroacetamide (MTBSTFA) was developed for the analysis of 19 chlorophenols compounds in atmospheric samples (gas and particles). Air sampling was carried out using a Hi-Vol sampler with glass fibre filter and XAD-2 resin at a flow rate of 60 m3 h−1. The particle and gas phases were collected separately over a period of 4 h. Samples were Soxhlet extracted, evaporated to dryness under nitrogen and refilled with acetonitrile. 100 mL of these extracts were derivatized with 100 μL of MTBSTFA at 80 °C for 1 h under strong stirring. Sylylated chlorophenols were injected into a GC-MS in splitless mode and quantified as their TBDMS derivatives in the SIM mode. Mass spectral analysis of the derivatives of the 19 compounds studied indicates that the spectra are highly specific showing an ion at [M - 57]+ which is useful for structure confirmation or analysis at low levels using selected ion monitoring. Quantification limits varied between 5 µg L−1 and 10 µg L−1 which correspond to 20 pg m−3 and 40 pg m−3 for 250 m3 of air sampled. This method was successfully applied to atmospheric samples collected simultaneously in winter 2004 in an urban (Strasbourg) and rural (Erstein) areas in east of France.

Analysis of Benzidine and Dichlorobenzidine at Trace Levels in Water by Silylation and Gas Chromatography–Mass Spectrometry

Conditions for silylation of benzidine (BZ) and 3,3′-dichlorobenzidine (DCBZ) have been optimized. Reactivity, repeatability, and derivative stability were compared for the silylating reagents N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBDMSTFA) and the catalysts 3% trimethylsilylimidazole (TMS-I) and 0.3% NH4-I–dithioerythritol. The results showed that derivatization with MTBDMSTFA/NH4-I containing 0.1 mg dithioerythritol was superior to other methods. The silylation conditions selected were reaction with (MTBDMSTFA)–NH4I, 1000:3, with catalysis by dithioerythritol, at 80 °C for 80 min. The TBDMS derivatives of BZ and DCBZ had very good chromatographic properties and very sensitive detection was achieved by gas chromatography with electron-impact ionization mass spectrometry (GC-EIMS). Simultaneous determination of BZ and DCBZ in water was developed on the basis of the TBDMS derivatives. Deuterated BZ (d8-BZ) was chosen as internal standard (IS) for analysis of water samples. BZ and DCBZ were extracted from water at pH 8.5 with dichloromethane and the extract was then dried and silylated. Recoveries of BZ and DCBZ were approximately 102 and 103% at a concentration of 2.0 ng mL−1. The coefficients of variation for BZ and DCBZ were less than 9 and 4% at concentrations of 0.2 and 0.5 ng mL−1, respectively. The method detection limits for 200 mL water were 0.004 ng mL−1 for BZ and 0.02 ng mL−1 for DCBZ.

SbCl5—Wet Acetonitrile: A New System for Chemoselective O‐Desilylation.

AbstractFor Abstract see ChemInform Abstract in Full Text.

A multiresidue method for the analysis of phenols and nitrophenols in the atmosphere

A method using GC-MS and derivatization with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was developed for the analysis of 20 phenolic compounds in atmospheric samples (gas and particles). Air sampling was carried out using a Hi-Vol sampler with glass fibre filter and XAD-2 resin at a flow rate of 60 m(3) h(-1). The particle and gas phases were collected separately over a period of 4 h. Samples were Soxhlet extracted, evaporated to dryness under nitrogen and refilled with acetonitrile. 100 microl of these extracts were derivatized with 100 microl of MTBSTFA at 80 degrees C for 1 h under strong stirring. Phenolic compounds were injected into a GC-MS in splitless mode and quantified as their TBDMS derivatives in the SIM mode. Mass spectral analysis of the derivatives of the 20 compounds studied indicates that the spectra are highly specific showing an ion at [M - 57]+ which is useful for structure confirmation or analysis at low levels using selected ion monitoring. Quantification limits varied between 5 microg l(-1) and 10 microg l(-1) which correspond to 20 pg m(-3) and 40 pg m(-3) for 250 m(3) of air sampled. This method was successfully applied to atmospheric samples.

SbCl 5–wet acetonitrile: a new system for chemoselective O-desilylation

A new efficient method for deprotection of TBDMS derivatives of phenols, primary alcohols, carboxylic acids and secondary amines, consisting of SbCl 5 and MeCN with 0.1% water (w/v), is reported. It effects inter alia desilylation of a CH 2OTBDMS group in the presence of a ketal function.

Analysis of 13C-mixed Triacylglycerol in Stool by Bulk (Ea-IRMS) and Compound Specific (GC/MS) Methods

This paper was presented in poster form at the 17th International Congress of Nutrition, August 27-31, Vienna. Austria (Annals of Nutrition & Metabolism 2001; 45(Suppl.1):349). Some of the data were also presented in poster form at the British Society of Gastroenterology Meeting, March 18-21, Glasgow, UK (Gut 2001; 48(Suppl.1):A91). The 13C-mixed triacylglycerol (MTG) breath test is used to measure intraluminal fat digestion. In normal digestion. 20–40% of the ingested 13C label is recovered in breath CO2. We aimed to identify the proportions of ingested label excreted in stool, as well as breath following ingestion of 13C-MTG by children with impaired exocrine pancreatic function and healthy controls. 13C enrichment of breath samples was measured by continuous flow isotope ratio mass spectrometry (IRMS) and cumulative percent dose recovered (cPDR) in 10 h was calculated. Total 13C of a faecal fat extract from each stool was measured by elemental analyser-IRMS, and 13C enrichment and concentration of the TBDMS derivative of octanoic acid was measured by GC/MS after hydrolysis of the fat extract. Stool 5-day cPDR was calculated. Mean breath cPDR was 35%. Mean cPDR in stool by combustion-IRMS and GC/ MS, respectively, was 0.8% and 1.0%. Therefore, the remaining 64% of the 13C label must remain in the body and variability in breath cPDR is due to postabsorptive rather than predigestive factors.

Determination of Blood Folate Using Acid Extraction and Internally Standardized Gas Chromatography–Mass Spectrometry Detection

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [13C6]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [13C6]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.

A sensitive endocannabinoid assay. The simultaneous analysis of N-acylethanolamines and 2-monoacylglycerols

Mammalian cells produce both N-arachidonoylethanolamine (20:4n-6 NAE, anandamide) and 2-arachidonoylglycerol (2-AG), lipid signaling molecules that activate cannabinoid receptors. Because both agonists occur in the presence of receptor-inactive congeners, we have developed a sensitive method for the simultaneous assay of N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAG). These lipid classes are isolated from total lipids by solid phase extraction and converted to tert-butyldimethylsilyl ( tBDMS) derivatives in the presence of deuterated analogs. The tBDMS derivatives are analyzed by gas chromatography/mass spectrometry using selected ion monitoring programs specific for NAE and 2-MAG. Individual NAEs and 2-MAGs can be quantified in the nanogram and subnanogram range. The NAE and 2-MAG compositions of rat organs and cultured JB6 cells are reported.

Analysis of gabapentin in serum and plasma by solid-phase extraction and gas chromatography-mass spectrometry for therapeutic drug monitoring.

A simple method for the determination of gabapentin (Neurontin) is described. The method uses solid-phase extraction by disk column and derivatization followed by gas chromatographic-mass spectrometric analysis. The single-step derivatization with MTBSTFA produces a t-BDMS derivative of both the carboxylic and amine moieties of the molecule. Each step of the procedure was optimized to assure reliable performance of the method. The assay limit of detection was 0.1 microg/mL with a linear range from 1.0 to 35 microg/mL. Within-run (n = 3) and between-run (n = 40) coefficients of variation were less than 8.2 and 15.9%, respectively. The method has proven reliable in routine production for more than a year, producing clean chromatography with unique ion fragments, consistent ion mass ratios, and no interferences. Statistical analysis of the gabapentin concentrations measured in 1020 random specimens over a 2-month period showed a mean concentration of 6.07 microg/mL with a standard deviation of 5.28.

Highly Functionalized Cyclohexenyl Systems: Enzymatic Resolution and Selective Oxirane Opening Reactions of p-Benzoquinone Derivatives

Diol 2, derived in two steps from p-benzoquinone, was converted to epoxy alcohol 3. The latter was resolved with crude Candida rugosa lipase in isopropenyl acetate/toluene to give acetate (−)-4 and residual alcohol (+)-3. Oxirane ring opening reactions of 3 and the corresponding acetate and TBDMS derivatives with a variety of nucleophiles gave diastereomerically pure 3,4,5,6-tetrasubstituted cyclohexene derivatives of type 6. X-ray crystal structures were obtained on compounds 6e, 6m and the diacetate prepared from 6l. Nitrogen nucleophiles provided entry in various aziridines 10. Pd(0)-catalyzed nucleophilic additions to substrate 5 cleanly provided the diastereomeric pattern of type 11. trans-5,6-Disubstituted cyclohexadienes 13 were obtained by an unusual syn-reductive elimination on bromocarbonates 12 utilizing Pd or Zn.

Arginine (Arg) Turnover and Oxidation in Neonates Receiving Total Parenteral Nutrition (TPN) • 1497

In human, Arg is generally considered a dispensable amino acid. However, it has been proposed that Arg may be an indispensable amino acid in neonates after an increase in blood ammonia level was observed when arginine was removed from amino acid solution used in parenteral nutrition.Method: In order to estimate net de novo synthesis, in vivo, in neonates fed parenterally, I evaluated Arg and citrulline (Cit) metabolism measuring respective plasma amino acids fluxes and whole-body rate of Arg oxidation based on a single pool model (Beaumier, 1995). Rate of conversion of plasma Cit to Arg was also measured using the Clarke and Bier equation (1982). Protein turnover was also measured usingα-ketoisocaproic acid (α-KIC) as representative metabolite of leucine metabolism (Matthews 1982). Experiments: After being on TPN for 4-5 days, providing 3 gm of protein/kg/day and a minimum of 120 kcal/kg/day, a known primed constant infusion of [5-13C] Arg,[15N2-guanidino] Arg, [5,5-2H2, 13C-ureido] Cit and [5,5,5-2H2] leucine was given for five hours. Blood and breath samples were taken once before and twice, at 30 minute intervals, at the end of the infusion. Expired 13CO2 was corrected for under-recovery of 13C-label from infused [5-13C] ornithine. Isotopic abundance of amino acids and α-KIC was measured by GC/MS as methyl ester-TFA and t-BDMS derivatives, respectively. Results: Under these conditions, plasma Arg and Cit flux were measured at 209 ± 17 and 37± 3 μmol/kg/h, respectively. Arg oxidation was measured at 76± 6 with a dietary Arg intake was 69 ± 10 μmol/kg/h. Rate of conversion of plasma Cit into Arg was 15 ± 3 μmol/kg/h. The small excess in Arg oxidation and the low rate of conversion support the concept that neonates have a very limited rate of Arg de novo synthesis. Leucine appearance from protein breakdown was 141 ± 18 μmol/kg/h, which represents 9.4 ± 1.2 g/kg/day of protein. Conclusion: Net rate of endogenous Arg synthesis is measurable in neonates receiving TPN but remains relatively small in comparison to the whole-body turnover of the amino acid. These results are consistent with a limiting capacity for neonates to synthesize arginine supporting the recommendation that Arg is an indispensable amino acid in neonates fed a parenteral solution.