Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography—mass spectrometry

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E₄ (LTE₄). Oxygen-18 labelling of LTE₄ methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE₄ to the extent of 89.8% ([¹⁸O₂]LTE₄) and one oxygen-18 atom to the extent of 9.4% ([¹⁶O¹⁸O]LTE₄), with only 0.7% remaining unchanged ([¹⁶O₂]LTE₄). [¹⁸O₂]LTE₄ was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4°C for up to 20 h. [¹⁸O₂]LTE₄ was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE₄ in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE₄ in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.     

Synthesis of homoagmatine and GC–MS analysis of tissue homoagmatine and agmatine: evidence that homoagmatine but not agmatine is a metabolite of pharmacological L-homoarginine in the anesthetized rat

Amino Acids - Low L-homoarginine (hArg) concentrations in human blood and urine are associated with renal and cardiovascular morbidity and mortality, yet the underlying mechanisms and the...     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

The Effects of Lung Protective Ventilation or Hypercapnic Acidosis on Gas Exchange and Lung Injury in Surfactant Deficient Rabbits

Background

Permissive hypercapnia has been shown to reduce lung injury in subjects with surfactant deficiency. Experimental studies suggest that hypercapnic acidosis by itself rather than decreased tidal volume may be a key protective factor.

Objectives

To study the differential effects of a lung protective ventilatory strategy or hypercapnic acidosis on gas exchange, hemodynamics and lung injury in an animal model of surfactant deficiency.

Methods

30 anesthetized, surfactant-depleted rabbits were mechanically ventilated (FiO₂ = 0.8, PEEP = 7cmH₂O) and randomized into three groups: Normoventilation-Normocapnia (NN)-group: tidal volume (Vt) = 7.5 ml/kg, target PaCO₂ = 40 mmHg; Normoventilation-Hypercapnia (NH)-group: Vt = 7.5 ml/kg, target PaCO₂ = 80 mmHg by increasing FiCO₂; and a Hypoventilation-Hypercapnia (HH)-group: Vt = 4.5 ml/kg, target PaCO₂ = 80 mmHg. Plasma lactate and interleukin (IL)-8 were measured every 2 h. Animals were sacrificed after 6 h to perform bronchoalveolar lavage (BAL), to measure lung wet-to-dry weight, lung tissue IL-8, and to obtain lung histology.

Results

PaO₂ was significantly higher in the HH-group compared to the NN-group (p<0.05), with values of the NH-group between the HH- and NN-groups. Other markers of lung injury (wet-dry-weight, BAL-Protein, histology-score, plasma-IL-8 and lung tissue IL-8) resulted in significantly lower values for the HH-group compared to the NN-group and trends for the NH-group towards lower values compared to the NN-group. Lactate was significantly lower in both hypercapnia groups compared to the NN-group.

Conclusion

Whereas hypercapnic acidosis may have some beneficial effects, a significant effect on lung injury and systemic inflammatory response is dependent upon a lower tidal volume rather than resultant arterial CO₂ tensions and pH alone.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Accurate quantification of dimethylamine (DMA) in human plasma and serum by GC-MS and GC-tandem MS as pentafluorobenzamide derivative in the positive-ion chemical ionization mode

Dimethylamine (DMA) circulates in human blood and is excreted in the urine. Major precursor for endogenous DMA is asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is hydrolyzed to DMA and L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH). In previous work, we reported a GC-MS method for the quantification of DMA in human urine. This method involves simultaneous derivatization of endogenous DMA and the internal standard (CD(3))(2)NH by pentafluorobenzoyl chloride (PFBoylCl) and extraction of the pentafluorobenzamide derivatives by toluene. In the present work, we optimized this derivatization/extraction procedure for the quantitative determination of DMA in human plasma. Optimized experimental parameters included vortex time and concentration of PFBoylCl, carbonate and internal standard. The GC-MS method was thoroughly validated and applied to measure DMA concentrations in human plasma and serum samples. GC-MS quantification was performed by selected-ion monitoring of the protonated molecules at m/z 240 for DMA and m/z 246 for (CD(3))(2)NH in the positive-ion chemical ionization mode. Circulating DMA concentration in healthy young women (n=18) was determined to be 1.43+/-0.23 micaroM in serum, 1.73+/-0.17 microM in lithium heparin plasma, and 9.84+/-1.43 microM in EDTA plasma. DMA was identified as an abundant contaminant in EDTA vacutainer tubes (9.3+/-1.9 nmol/monovette, n=6). Serum and lithium heparin vacutainer tubes contained considerably smaller amounts of DMA (0.42+/-0.01 and 0.95+/-0.01 nmol/monovette, respectively, each n=6). Serum is recommended as the most appropriate matrix for measuring DMA in human blood. The present GC-MS method should be useful for the determination of systemic and whole body DDAH activity by measuring circulating and excretory DMA in experimental and clinical studies.     

Gas chromatographic-tandem mass spectrometric quantification of free 3-nitrotyrosine in human plasma at the basal state

A fully validated gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate and precise quantification of free 3-nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4-4.2 nM) for free 3-nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-nitrotyrosine and 3-nitro-l-[(2)H(3)]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-microl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester-pentafluoropropionyl amide-trimethylsilyl ether derivatives, and quantified by GC-tandem MS. Overall recovery was determined as 50 +/- 5% using 3-nitro-l-[(14)C(9)]tyrosine. The limit of detection of the method was 4 amol of 3-nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[(14)C(9)]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of > or = 80% and a precision of > or = 94%. The method should be useful to investigate the utility of plasma free 3-nitrotyrosine as an indicator of nitric oxide ((.)NO)-associated oxidative stress in vivo in humans.     

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). I. Direct evidence for cis-EODA formation from oleic acid oxidation by liver microsomes and isolated hepatocytes

Oleic acid, cis-9-octadecenoic acid, is the major fatty acid in mammals. Its oxide, cis-9,10-epoxyoctadecanoic acid (cis-EODA), has been identified in blood and urine of humans, its origin is, however, still unknown. Lipid peroxidation and enzyme-catalyzed epoxidation of oleic acid are two possible sources. In the present article, we investigated by HPLC and GC-MS whether cis-EODA is formed enzymatically from oleic acid by the cytochrome P450 (CYP) system. Oleic acid, cis-EODA and its hydratation product threo-9,10-dihydroxyoctadecanoic acid (threo-DiHODA) were quantitated by HPLC as their p-bromophenacyl esters. For structure elucidation by GC-MS, the pentafluorobenzyl (PFB) esters of these compounds were isolated by HPLC and converted to their trimethylsilyl ether derivatives. Liver microsomes of rats, rabbits and humans oxidized oleic acid into cis-EODA. This is the first direct evidence for the enzymatic formation of cis-EODA from oleic acid. The epoxidation of oleic acid was found to depend on CYP, NADPH+H(+), and O(2). cis-EODA was measurable in incubates of liver microsomes for up to 30 min of incubation. Maximum cis-EODA concentrations were reached after 5-7 min of incubation and found to depend upon oleic acid concentration. Isolated rat hepatocytes hydratated cis-EODA into threo-DiHODA which was further converted to unknown metabolites. However, from incubation of oleic acid with these cells we could not detect threo-DiHODA or cis-EODA. Our study suggests that circulating and excretory cis-EODA may originate, at least in part, from CYP-catalyzed epoxidation of oleic acid. GC-MS of intact cis-EODA as its PFB ester in the negative-ion chemical ionization mode should be useful in investigating the physiological role of cis-EODA in man.     

Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.     

Accurate quantification of basal plasma levels of 3-nitrotyrosine and 3-nitrotyrosinoalbumin by gas chromatography-tandem mass spectrometry

Measurement of 3-nitro-L-tyrosine (NO(2)Tyr) and protein-related 3-nitro-L-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC-tandem MS-based method for NO(2)Tyr which yielded the lowest basal plasma NO(2)Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO(2)TyrALB) in human plasma. NO(2)TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- L-[2H(3)]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO(2)TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO(2)TyrALB/TyrALB, was determined to be 1.55+/-0.54x1:10(6) (mean+/-SD). The plasma concentration of NO(2)TyrALB was estimated as 24+/-4 nM. The NO(2)Tyr plasma levels in these volunteers were determined to be 0.73+/-0.53 nM. In the same volunteers, NO(2)TyrALB/TyrALB, NO(2)TyrALB and NO(2)Tyr were measured 15 days later and the corresponding values were determined to be 1.25+/-0.58x1:10(6), 25+/-6 nM and 0.69+/-0.16 nM. For comparison, NO(2)Tyr and NO(2)TyrALB were measured in six plasma samples from healthy volunteers by GC-MS and GC-tandem MS. Different values were found for NO(2)Tyr, i.e. 5.4+/-2.8 versus 2.7+/-1.5 nM, and comparable values for NO(2)TyrALB/TyrALB, i.e. 0.5+/-0.2x1:10(6) versus 0.4+/-0.1x1:10(6), by these methods. The ratio of the values measured by GC-MS to those measured by GC-tandem MS were 2.9+/-3.1 for NO(2)Tyr and 1.2+/-0.2 for NO(2)TyrALB/TyrALB. The present GC-tandem MS method provides accurate values of NO(2)Tyr and NO(2)TyrALB in human plasma.