Comparative pharmacokinetics of seven bioactive components in normal, sham-operated, and myocardial ischemia-reperfusion injury rats after oral administration of the Salvia Miltiorrhiza-Moutan Cortex herb pair.

Recently the Salvia Miltiorrhiza-Moutan Cortex (SM-MC) herb pair is considered as a promising Chinese medicinal mixture exhibiting a range of pharmacological activities, including treating cardiovascular disease due to its unique composition. In this study, we conducted the comparative pharmacokinetic analysis of seven main bioactive components of SM-MC in a different model rat. A straightforward ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) strategy that could simultaneously evaluate the levels of seven compounds was used to ensure the reliability of these pharmacokinetic analyses in rat plasma. The rat plasma samples were collected from normal, sham-operated, and myocardial ischemia-reperfusion injury (MIRI) groups at predetermined time points after the administration of SM-MC. The main pharmacokinetic parameters were detected and calculated. We successfully assessed the maximum concentration (Cmax ), time to Cmax (Tmax ), the elimination rate constant (λz ), total half-life (t1/2 ), total body clearance (CL), and the area under the concentration-time curve from 0 to last sampling time (AUC0-t ) and extrapolated to infinity (AUC0-∞ ). To sum up, an optimized UPLC-MS/MS approach that could be used to rapidly, simultaneously, and sensitively detect seven bioactive compounds derived from SM-MC extract preparations was successfully developed, which may offer a pharmacokinetic basis for preclinical and clinical studies of SM-MC herb pair for treating MIRI.

Simultaneous determination of eight bioactive components of Qishen Yiqi dripping pills in rat plasma using UFLC-MS/MS and its application to a pharmacokinetic study.

In this study, a rapid and reliable ultra-fast liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of eight active ingredients, including astragaloside IV, ononin, tanshinol, protocatechualdehyde, protocatechuic acid, salvianolic acid D, rosmarinic acid and ginsenoside Rg1 , in rat plasma. The plasma samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was performed on a Waters Acquity UPLC® BEH C18 column (1.7 µm particles, 2.1 × 100 mm). The mobile phase consisted of 0.1% aqueous formic acid (A)-acetonitrile with 0.1% formic acid (B) at a flow rate of 0.4 mL/min. Quantification was performed on a triple quadruple tandem mass spectrometry with electrospray ionization by multiple reaction monitoring both in the negative and in the positive ion mode. The lower limit of quantification of tanshinol was 2.0 ng/mL and the others were 5.0 ng/mL. The extraction recoveries, matrix effects, intra- and inter-day precision and accuracy of eight tested components were all within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of the eight active constituents after intragastric administration of three doses (1.0, 3.0, 6.0 g/kg body weight) of Qishen Yiqi Dripping Pills to rats.

Simultaneous determination and pharmacokinetic study of four phenolic acids in rat plasma using UFLC-MS/MS after intravenous administration of salvianolic acid for injection.

A simple, sensitive and selective ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was established for simultaneous determination and pharmacokinetic study of rosmarinic acid (RA), salvianolic acid D (Sal D), lithospermic acid (LA) and salvianolic acid B (Sal B) in rat plasma after intravenous administration of salvianolic acid for injection (SAFI). Three doses of administration, containing 14, 28 and 56mg/kg, were investigated in this study. Plasma samples were pretreated using protein precipitation (PP) with pre-cooled acetonitrile. Chromatographic separation was achieved on a CORTECS™ UPLC C18 column (1.6µm, 2.1×100mm) with a mobile phase composed of 0.1% formic acid aqueous (V/V) and 0.1% formic acid acetonitrile (V/V). Analytes were detected using electrospray ionization (ESI) source in negative ionization mode and quantified in multiple reaction monitoring (MRM) mode. The validated method is stable and reliable. No significant difference of half lives (t1/2) of four analytes at three doses was observed. Area under the curve (AUC0-∞) and peak concentration (Cmax) of the four analytes demonstrated a linear increase in across the doses with the linear correlation r of each analyte at three doses were greater than 0.95. It indicated that the pharmacokinetic behavior of SAFI is positively related to dose at the range of 14-56mg/kg.

Development and validation of a UFLC-MS/MS method for determination of 7'(Z)-(8″S, 8‴S)-epi-salvianolic acid E, (7'R, 8'R, 8″S, 8‴S)-epi-salvianolic acid B and salvianolic acid B in rat plasma and its application to pharmacokinetic studies.

7'(Z)-(8″S, 8‴S)-epi-Salvianolic acid E (compound 1) and (7'R, 8'R, 8″S, 8‴S)-epi-salvianolic acid B (compound 2), two novel analogs of salvianolic acid B (Sal B), have been recently isolated from Salvianolic acid for injection. They both show powerful antioxidant effects, including inducing NQO1 activity and scavenging DPPH free radical, and potential protecting effects for cerebral ischemia. However, no reports have been described the pharmacokinetic study of them. In this study, an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for the determination of compound 1, compound 2 and Sal B in rat plasma, respectively. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters Acquity UPLC(®) HSS T3 column (1.7µm particles, 2.1mm i.d.×100mm) with the mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B) (65:35, v/v). Quantification was performed on a triple quadruple tandem mass spectrometry with electrospray ionization (ESI) by multiple reaction monitoring (MRM) in the negative ion mode. Monitored transitions were set at m/z 717.0→519.0, 717.1→519.1, 717.2→518.9 and 320.9→152.1 for compound 1, compound 2, Sal B and chloramphenicol (internal standard, IS), respectively. Linear calibration curves were acquired over the concentration range of 2.0-1000ng/mL for the three analytes in rat plasma. The extraction recoveries, matrix effects, intra- and inter-day precisions and accuracies of the three analytes were all within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of compound 1, compound 2 and Sal B after intravenous administration of 6.0mg/kg in rats, respectively. The results indicated that compound 1 and compound 2 were both eliminated more slowly than Sal B. Exposure levels of both compound 1 and Sal B were higher than compound 2 in the same dosage range. This study provided critical reference for the pharmacokinetic study of compound 1 and compound 2.

Determination of salvianolic acid C in rat plasma using liquid chromatography-mass spectrometry and its application to pharmacokinetic study.

A sensitive and reliable LC-ESI-MS method for the determination of salvianolic acid C in rat plasma has been developed and validated. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate and separated on a Zorbax SB-C18 column (3.5 µm, 2.1 × 100 mm) at a flow rate of 0.3 mL/min using acetonitrile-water as mobile phase. The detection was carried out by a single quadrupole mass spectrometer with electrospray ionization source and selected ion monitoring mode. Linearity was obtained for salvianolic acid C ranging from 5 to 1000 ng/mL. The intra- and inter-day precisions (RSD, %) didn't exceed 9.96%, and the accuracy (RE, %) were all within ±3.64%. The average recoveries of the analyte and internal standard were >89.13%. Salvianolic acid C was proved to be stable during all sample storage, preparation and analytic procedures. The validated method was successfully applied to pharmacokinetic study after oral and intravenous administration of salvianolic acid C to rats. The absolute oral bioavailability of salvianolic acid C was 0.29 ± 0.05%. This method was further applied to simultaneous determination of salvianolic acid A, salvianolic acid B and salvianolic acid C in rat plasma and showed good practicability.

[BRAIN FOCAL ISCHEMIA-REPERFUSION CAUSES A DECREASED RESISTANCE OF ERYTHROCYTES FROM VENOUS BLOOD TO ACID HEMOLYSIS, WHICH IS PREVENTED BY ECDYSTERONE].

We investigated the resistance of erythrocytes from rat brain venous blood to acid hemolysis in the dynamics of brain ischemic period (15, 30, 45 and 60 min), as well as in the early (5 min) and distant (24h) period of brain reperfusion. Brain ischemia-reperfusion was made in rats that received ecdysterone (standartized extract of Serratula coronata) within 18 days (per os, 1 mg/kg). Analysis of the kinetic curves of acid hemolysis showed a pronounced (60 times, from 1.45 to 85.85% at 60 min of brain ischemia and at 5 min of brain reperfusion, respectively) increase of unstable erythrocytes that hemolyzed easily (< 2.5 min). In the preconditioned rats, this increase was only 8-fold. During the period of brain ischemia, with a maximum at 15th minute, in the venous blood from brain the diene conjugates (DK) pools increased from 2.40 to 9.48 ng/mg protein and LTC4 pools increased from 1.49 to 5.98 pmol/mg protein. Even more pools of DC and LTC4 were increased at 5th min of brain reperfusion. In animals received ecdysterone, during ischemia and early reperfusion period, both pools of DC and LTC4 in venous blood were lower than that in the controls. The latter implies a possible antiradical mechanism of the protective effect of ecdysterone.

Human blood and plasma partition coefficients for C4-C8 n-alkanes, isoalkanes, and 1-alkenes.

Human blood:air and plasma:air partition coefficients for C(4)-C(8) n-alkanes, isoalkanes, and 1-alkenes were determined using multiple headspace extraction coupled with solid phase microextraction and gas chromatography. Mean blood:air partition coefficients expressed in the form of dimensionless blood-to-air concentration ratio (g/mL(b)/g/mL(a)) were 0.183, 0.416, 1.08, 2.71, and 5.77 for C(4)-C(8) n-alkanes; 0.079, 0.184, 0.473, 1.3, and 3.18 for C(4)-C(8) isoalkanes; and 0.304, 0.589, 1.32, 3.5, and 7.01 for C(4)-C(8) 1-alkenes, respectively (n = 8). The reported partition coefficient values increased exponentially with boiling points, molecular weights, and the carbon atoms in the particle. The solubility of 1-alkenes in blood was higher than in plasma, whereas the blood:air and plasma:air partition coefficients of n-alkanes and isoalkanes did not differ significantly. Consequently, additional interactions of 1-alkenes with whole blood seem to occur. The presented findings are expected to be particularly useful for assessing the uptake, distribution, and elimination of hydrocarbons in human organism.

Novel methods for the quantification of (2E)-hexadecenal by liquid chromatography with detection by either ESI QTOF tandem mass spectrometry or fluorescence measurement.

Sphingosine-1-phosphate lyase (SPL) is the only known enzyme that irreversibly cleaves sphingosine-1-phosphate (S1P) into phosphoethanolamine and (2E)-hexadecenal during the final step of sphingolipid catabolism. Because S1P is involved in a wide range of physiological and diseased processes, determining the activity of the degrading enzyme is of great interest. Therefore, we developed two procedures based on liquid chromatography (LC) for analysing (2E)-hexadecenal, which is one of the two S1P degradation products. After separation, two different quantification methods were performed, tandem mass spectrometry (MS) and fluorescence detection. However, (2E)-hexadecenal as a long-chain aldehyde is not ionisable by electrospray ionisation (ESI) for MS quantification and has an insufficient number of corresponding double bonds for fluorescence detection. Therefore, we investigated 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) as a derivatisation reagent. DAIH transforms the aldehyde into an ionisable and fluorescent analogue for quantitative analysis. Our conditions were optimised to obtain the outstanding limit of detection (LOD) of 1 fmol per sample (30 µL) for LC-MS/MS and 0.75 pmol per sample (200 µL) for LC determination with fluorescence detection. We developed an extraction procedure to separate and concentrate (2E)-hexadecenal from biological samples for these measurements. To confirm our new methods, we analysed the (2E)-hexadecenal level of different cell lines and human plasma for the first time ever. Furthermore, we treated HT-29 cells with different concentrations of 4-deoxypyridoxine (DOP), which competitively inhibits pyridoxal-5-phosphate (P5P), an essential cofactor for SPL activity, and observed a significant decrease in (2E)-hexadecenal relative to the untreated cells.

Picogram measurement of volatile n-alkanes (n-hexane through n-dodecane) in blood using solid-phase microextraction to assess nonoccupational petroleum-based fuel exposure.

We describe here a new method for the analysis of alkanes ( n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, and n-dodecane) in blood using headspace solid-phase microextraction gas chromatography/mass spectrometry. This method is used to measure picogram per milliliter levels of n-alkanes in blood that may result from nonoccupational exposure to alkanes and other volatile nonpolar compounds from common sources such as petroleum-based fuel. This alkane signature is useful in distinguishing typical fuel biomarkers (e.g., benzene and toluene) from other confounding exposure sources such as cigarette smoke. Development of this method required special attention to sample handling as alkanes are not highly soluble in aqueous matrixes and exist as ubiquitous compounds found in many laboratory materials and the environment. In particular, significant n-hexane contamination ( approximately 0.4 ng/mL) occurred from collecting blood samples in vacutainers. This residue was removed by boiling the vacutainer stoppers in methanol followed by vacuum baking. For all the alkanes, the calculated accuracy demonstrated for the water-based standards ranged from 3.3% to 17% as deduced from the difference of the lowest and middle standards from the curve fit. Quality control data among runs over a 10 month period were found to vary from 14% to -29%, with a few exceptions. The resulting quantification limits for n-hexane through n-decane ranged from 0.069 to 0.132 ng/mL. In the analysis of 1200 blood samples from people with no known occupational exposure, median blood levels for all n-alkanes were below these quantification limits. n-Hexane levels above the method detection limit were, however, found in 1.3% of the samples.

Pro- and antioxidants in the central lymph in experimental chronic toxic hepatitis.

Using rat model of chronic toxic hepatitis we showed the involvement of the lymph system in the formation of the response to toxic liver damage consisting in deposition of LPO products in the central lymph and active involvement of antioxidants in their neutralization with redistribution of ceruloplasmin into lymph vessels against the background of reduced a-tocopherol content in the central lymph.

Low nitric oxide bioavailability contributes to the genesis of experimental cerebral malaria.

The role of nitric oxide (NO) in the genesis of cerebral malaria is controversial. Most investigators propose that the unfortunate consequence of the high concentrations of NO produced to kill the parasite is the development of cerebral malaria. Here we have tested this high NO bioavailability hypothesis in the setting of experimental cerebral malaria (ECM), but find instead that low NO bioavailability contributes to the genesis of ECM. Specifically, mice deficient in vascular NO synthase showed parasitemia and mortality similar to that observed in control mice. Exogenous NO did not affect parasitemia but provided marked protection against ECM; in fact, mice treated with exogenous NO were clinically indistinguishable from uninfected mice at a stage when control infected mice were moribund. Administration of exogenous NO restored NO-mediated signaling in the brain, decreased proinflammatory biomarkers in the blood, and markedly reduced vascular leak and petechial hemorrhage into the brain. Low NO bioavailability in the vasculature during ECM was caused in part by an increase in NO-scavenging free hemoglobin in the blood, by hypoargininemia, and by low blood and erythrocyte nitrite concentrations. Exogenous NO inactivated NO-scavenging free hemoglobin in the plasma and restored nitrite to concentrations observed in uninfected mice. We therefore conclude that low rather than high NO bioavailability contributes to the genesis of ECM.

Circulating 4-hydroxynonenal-protein thioether adducts assessed by gas chromatography-mass spectrometry are increased with disease progression and aging in spontaneously hypertensive rats.

Oxidative stress has been implicated in numerous degenerative diseases of aging, including heart diseases. However, there is still a need to identify biomarkers of oxidative stress-related events, such as protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in these diseases in humans. The objective of this study was to assess if circulating levels of HNE-protein adducts (i) can be assessed with precision by GCMS and (ii) vary with disease progression and aging in a model of cardiomyopathy that displays enhanced oxidative stress, namely the spontaneously hypertensive rats (SHR). We modified a previously published isotope dilution GCMS method that quantifies HNE and its inactive metabolite, 1,4-dihydroxynonene (DHN), bound to thiol proteins following treatment with NaB(2)H(4) and Raney nickel, to increase its sensitivity (20-fold), precision, and robustness. Levels of these adducts were measured in blood and plasma collected from SHR and control Wistar rats at 7, 15, 22, and 30 weeks of age. Levels of protein-bound HNE, which were quantitated with good precision in the nanomolar range in blood, but not in plasma, were significantly increased by disease (SHR) and age (P < 0.0001 for both). Compared to Wistar rats, SHR showed greater blood levels of HNE-protein adducts at 22 and 30 weeks. Levels of protein-bound DHN, which were detected in blood and in plasma, were not affected by disease or age. Collectively, the results of this study conducted in an animal model of cardiomyopathy demonstrate that changes in blood HNE-protein thioether adducts with disease progression and aging can be assessed with good precision by the described GCMS method. This method may prove to be useful in evaluating the occurrence and impact of oxidative stress-related events involving bioactive HNE in heart diseases and aging in humans.

Effects of metabolic control and vascular complications on indices of oxidative stress in type 2 diabetic patients.

The direct effect and the interaction of diabetic angiopathy and metabolic control on free radical and antioxidant activity indices was investigated in 48 patients with type 2 diabetes mellitus. Conjugated dienes (CD) and thiobarbituric acid-reacting substances (TBARS) levels were 34 and 178% of control values, respectively. An approximate two-fold decrease in plasma thiols (PSH) and erythrocyte lysate thiols (LSH) concentrations, parameters reflecting protein oxidative damage, was found. Impairment of blood antioxidant potential in diabetic patients was reflected by an 81% increase in superoxide dismutase (SOD) activity, a 30% decrease in catalase (CT), 20% decrease in glutathione peroxidase (GPx) and glutathione reductase (GR) activities as well as by lowered total antioxidant status (TAS). CD, TBARS and SOD values were positively correlated with plasma glucose concentration and glycated hemoglobin level. A negative correlation existed between levels of LSH, PSH, CT, GPx or TAS and both glucose and HbA(1c). Blood glucose control and vascular complications had strong independent effects on prooxidant-antioxidant status, apart from blood glucose and GR activity. In addition, glycemic control and diabetic vasculopathy interact in their influence on most of the free radical and antioxidant indices, except for CD, LSH levels and CT activity. Thus, we observed different mechanisms by which vascular complications and glucose control affect blood free radical indices and antioxidant status parameters in type 2 diabetic patients.

Comparison of the predicted in vivo behaviour of the Sn(II)-APDDMP complex and the results as studied in a rodent model.

In a quest for more effective radiopharmaceuticals for pain palliation of metastatic bone cancer, this paper relates results obtained with ((117m)Sn labelled) Sn(II) complexed to the bone seeking bisphosphonate, N,N-dimethylenephosphonate-1-hydroxy-3-aminopropylidenediphosphonate (APDDMP). APDDMP is synthesised from the known bone cancer pain palliation agent 1-hydroxy-3-aminopropylidenediphosphonate (APD, Pamindronate). This work is performed to utilise the idea that the low bone marrow radio toxicity of (117m)Sn could afford a highly effective radiopharmaceutical in pain palliation but also in the curative treatment of bone metastasis. Complex-formation constants of APDDMP with the important blood plasma metal-ions, Ca(2+), Mg(2+), Zn(2+) as well as the added metal ion, Sn(2+) were measured by glass electrode potentiometry at 25 degrees C and I = 150 mM. Blood plasma models were constructed using the computer code ECCLES and the results compared with those gathered from tests on a rodent model. The ((117m)Sn-labelled) Sn(II)-APDDMP complex was found to have only some liver and bone uptake although a high trabecular to normal bone ratio was recorded. From the blood plasma model this was shown to be primarily due to the high affinity of APDDMP for Ca(II) causing some of the Sn(II)-APDDMP complex to dissociate. High kidney uptake and excretion as well as high bladder uptake was recorded which was shown to be due to the dissociation of the Sn(II)-APDDMP complex in blood plasma. Animal model observations could be explained by the blood plasma modelling.

Design, synthesis, anti-HIV activities, and metabolic stabilities of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors.

The alkenyldiarylmethane (ADAM) HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are effective anti-HIV agents in cell culture. However, the potential clinical utility of the ADAMs is expected to be limited by the presence of methyl ester moieties that are likely to be metabolized by nonspecific esterases in blood plasma to biologically inactive carboxylic acid derivatives. The present investigation was therefore undertaken to investigate the anti-HIV activities of the ADAMs versus HIV-1(IIIB) and HIV-2(ROD) in MT-4 cells and the stabilities of the biologically active ADAMs in rat plasma. The ADAMs displayed a wide range of metabolic stabilities in rat plasma, with half-lives ranging from 0.9 to 76.6 min. A wide assortment of structural modifications was tolerated, with 18 of the 32 compounds tested displaying EC(50) values between 0.3 and 3.7 microM versus HIV-1(IIIB) in MT-4 cells, 3 compounds in the EC(50) = 13.2-35.4 microM range, and the remaining compounds inactive. Consistent with the mechanism of action of the ADAMs as NNRTIs, they were inactive or displayed comparatively low activity versus HIV-2(ROD). The replacement of the two aromatic methyl ester substituents in one of the most active ADAMs (EC(50) = 0.6 microM) with two methyl thioester groups resulted in an increase in plasma half-life from 5.8 to 55.3 min, while maintaining the antiviral potency at the EC(50) = 1.8 microM level. At the same time, the bis(thioester) modification was less cytotoxic to uninfected MT-4 cells, with a CC(50) of >224 microM versus 160 microM for the parent compound.

Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol.

Previous work has shown that the endogenous cystathionine gamma-synthase (CSE)/hydrogen sulfide (H(2)S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H(2)S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H(2)S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H(2)S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H(2)S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.

Assay of ezlopitant, a substance P receptor antagonist, and metabolites in biological matrices by gas chromatography with mass spectrometric detection: simultaneous analysis of a benzyl alcohol and alkene.

A method for the analysis of the substance P antagonist ezlopitant and two active metabolites in serum using solid-phase extraction followed by GC-MS analysis is described. The linear dynamic range was 1.0 to 100 ng/ml and precision and accuracy over this range were within 15%. Upon injection of reconstituted sample extracts into the hot injector port of the gas chromatograph, the benzyl alcohol metabolite undergoes a small amount of spontaneous dehydration to the alkene metabolite. We have incorporated an additional hexadeuterated internal standard of the benzyl alcohol into the assay to permit measurement of the extent of dehydration in each sample. This novel approach should be generally applicable to the simultaneous determination of benzyl alcohols and corresponding alkenes by GC-MS when the possibility exists that the alcohol can undergo spontaneous dehydration to the alkene in the injector port of GC instrumentation.

Identification of cuticular hydrocarbons and the alkene precursor to the pheromone in hemolymph of the female gypsy moth, Lymantria dispar.

Hydrocarbons were extracted from the surface of the cuticle and from the hemolymph of adult female gypsy moths. GC and GC/MS analysis indicated that the cuticular hydrocarbons with chain lengths >21 carbons were the same as those found in the hemolymph. These consisted of mostly saturated straight chain hydrocarbons with heptacosane the major component. Methyl branched hydrocarbons were also identified including a series of tetramethylalkanes with chain lengths of 30, 32, and 34 carbons. In addition to those found on the cuticle surface, the hemolymph contained the alkene pheromone precursor, 2-methyl-Z7-octadecene and two saturated analogues, 2-methyl-octadecane and 2-methyl-hexadecane. No evidence was obtained for the presence of the pheromone 2-methyl-7, 8-epoxy-octadecane in the hemolymph. Pheromone gland extracts indicated that small amounts (<1 ng) of the alkene precursor were also present in the gland. Relatively larger amounts of the alkene precursor were found in the hemolymph at the time when pheromone titers were higher on the gland. The presence of the hydrocarbon pheromone precursor in the hemolymph is discussed in relation to possible biosynthetic pathways for producing the gypsy moth pheromone.

Quantitation of monosaccharide isotopic enrichment in physiologic fluids by electron ionization or negative chemical ionization GC/MS using di-O-isopropylidene derivatives.

The aldonitrile pentaacetate and other derivatives lack ions in the electron ionization (EI) spectra possessing an intact hexose structure and thus must be analyzed by chemical ionization GC/MS in order to study multiple isotopomers. We report methods for quantitation of hexose di-O-isopropylidene acetate (IPAc) or pentafluorobenzoyl (PFBz) esters. These were prepared in a two-step procedure using inexpensive reagents that do not adversely impact the isotopomer structure of the sugar. The acetate derivative possesses an abundant [M - CH3] ion in the EI spectrum which is suitable for quantitative analysis of isotopomers. The negative chemical ionization (NCI) spectrum of the corresponding pentafluorobenzoyl derivative has a dominant molecular anion. Moreover, the PFBz derivative is about 100-fold more sensitive than the acetate, which offers some advantages for analysis of minor hexoses found in plasma. Isotopic calibration curves of [U-13C]glucose are linear over the 0.1-60% tracer/tracee range tested. The useful range for isotopic tracer studies is 25-2500 pmol for EI analysis of the acetate derivative and 0.1-55 pmol for NCI analysis of PFBz derivative (sample amount injected). For most studies where sample size is not limited, EI-GC/MS analysis of the IPAc derivative is preferred. NCI-GC/MS analysis is reserved when sample size is limiting or when studies involve hexoses other than glucose that are normally present at low concentration.

Cardiopulmonary bypass increases coronary IL-8 in diabetic patients without evidence of reperfusion injury.

BACKGROUND: Endothelin-1 (ET-1) has been shown to be a potent agonist for monocyte production of the neutrophil chemotactic cytokine interleukin-8 (IL-8). We have shown that diabetic patients demonstrate elevated coronary ET-1 after coronary artery bypass grafting (CABG). We hypothesized that these same diabetic patients would manifest elevated coronary IL-8 and conjugated diene concentrations (an index of reperfusion injury). METHODS: Sixteen patients [9 nondiabetics and 7 type II diabetics] underwent nonemergent CABG. The two groups did not differ significantly in preoperative ejection fraction, number of vessels bypassed, or cross-clamp time. Coronary sinus samples were obtained prior to cardioplegic arrest (baseline) and at 1 and 15 min after reperfusion periods A and B (A, reperfusion of native coronaries + LIMA; B, reperfusion of saphenous vein grafts in addition to native coronary system + LIMA). Plasma samples were analyzed for IL-8 (ELISA) and conjugated dienes (spectrophotometry). RESULTS: Initially after reperfusion, IL-8 in both groups was significantly lower than precardioplegia values. In reperfusion B, only the diabetic group demonstrated a significant increase in IL-8 concentrations at 1 and 15 min compared to nondiabetics. Conjugated diene levels were significantly higher in diabetics at each time point than nondiabetics. CONCLUSIONS: This study demonstrates an early decrease in IL-8 in both groups, most likely related to depressed production secondary to hypothermia. The subsequent elevation in IL-8 only in the diabetic group was seen without concomitant conjugated diene elevation. While no evidence of reperfusion injury was demonstrated in this time frame, the elevation of IL-8 in diabetics after CABG may contribute to later infiltration and associated oxidative damage.