Brain opiate receptor concentrations are increased in adult spontaneously hypertensive rats.

The saturable binding of 3H-naltrexone in the brains of eight week old spontaneously hypertensive rats (SHR) is about twice that measured in corresponding normotensive WKY rats. This increase is dependent on age since in three and four week old SHR and WKY rats no difference in binding is observed. Scatchard analysis of the saturation curves for the adult animals revealed that the change in binding is due to an increase in the number of binding sites and does not reflect a difference in binding affinity. The increase in opiate receptor content of SHR rats coincides with the appearance of elevated blood pressure in these animals, and supports a concept in which an interaction between the endorphins and the endocrine system may be involved in the mechanisms controlling hypertension.     

Atrial natriuretic peptides cleaved by endopeptidase are inactive in conscious spontaneously hypertensive rats

The dose-related natriuretic and depressor responses to atrial natriuretic peptides (ANP) 99-126, 103-126 and 103-123 were determined in unanesthetized spontaneously hypertensive rats (SHR) and were compared to the activities of their Cys105-Phe106 ring-opened metabolites. These metabolites were previously identified as the major initial products formed by incubation of the intact peptides with neutral endopeptidase (NEP). The areas over the curves (AOC) of the depressor responses to the intact peptides were dose-related and, at 30 nmole/kg, iv were greatest for ANP 99-126 and 103-126 (833 +/- 241 and 1157 +/- 221 mm Hg x min). Thirty nmole/kg of ANP 103-123, a possible product of NEP cleavage of ANP 103-126, produced a lesser AOC (442 +/- 152 mm Hg x min) than did either of the longer peptides. The AOC responses to 100 nmole/kg of the ring-opened metabolites of ANP 99-126, 103-126 and 103-123 (105 +/- 80, 153 +/- 43 and 148 +/- 64 mm Hg x min) were not significantly different from the effect of vehicle treatment (84 +/- 23 mm Hg x min). Although the natriuretic responses to increasing doses of the intact peptides did not occur in a linear fashion, sodium excretion was maximally elevated by 24 +/- 4, 16 +/- 3 and 10 +/- 3 microEq/kg/min by 3 nmole/kg of ANP 99-126, 30 nmole/kg of ANP 103-126 and 10 nmole/kg of ANP 103-123, respectively. In contrast, the natriuretic responses to 100 nmole/kg of the ring-opened metabolites of ANP 99-126, 103-126 and 103-123 (1 +/- 0, 5 +/- 2 and 2 +/- 1 microEq/kg/min, respectively) were not significantly different from the response to vehicle treatment (3 +/- 1 microEq/kg/min). In conclusion, three ring-opened products of NEP cleavage of ANP 99-126, 103-126 and 103-123 were inactive in conscious SHR.     

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 microgram of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Determination of 4-chloroindoleacetic acid methyl ester in Vicieae species by gas chromatography-mass spectrometry

The natural chlorinated auxin, 4-chloroindoleacetic acid methyl ester, was identified in immature seeds of Lathyrus sativus L., Lathyrus maritimus (L.) Bigel and Lathyrus odoratus L. by thin layer chromatography and gas chromatography-mass spectrometry. In immature seeds of Vicia sativa L. and Lens culinaris Medik. the hormone was identified by selected ion monitoring. The hormone was determined quantitatively using pentadeuterated 4-chloroindoleacetic acid methyl ester as internal standard. Contents varied from 1 mg/kg fresh weight in Lathyrus sativus to 0.02 mg/kg in Lens culinaris. Lathyrus maritimus also contained indoleacetic acid methyl ester (0.3 mg/kg) besides the chlorinated analogue.     

Quantitation of leukotriene B4 in human serum by negative ion gas chromatography-mass spectrometry

Leukotriene B4 (LTB4) is a potent chemotactic agent formed via the 5-lipoxygenase pathway from arachidonic acid. To understand the role LTB4 plays in several pathological processes it is essential that endogenous concentrations of LTB4 be accurately quantitated. We have developed a method based on electron capture negative ion mass spectrometry for the analysis of LTB4 in serum at low picogram per milliliter concentrations. Blood is collected into the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) to suppress ex vivo formation. Serum is isolated, equilibrated with the internal standard [2H4]LTB4, and extracted using octadecyl-silica (C-18) cartridges. After conversion of the carboxylic acids to their pentafluorobenzyl esters the extract is purified by straight-phase HPLC. Gas chromatographic-mass spectrometric analysis is accomplished on the tert-butyldimethylsilyl ether derivatives using dual-selected ion monitoring of m/z 431 and 435. These ions correspond to loss of tert-butyldimethylsilanol from the (M-PFB)- ion of endogenous and [2H4]LTB4, respectively. The concentration of LTB4 in human serum samples was 10.0 +/- 4.0 pg/ml (n = 5). The assay exhibited satisfactory precision, with an intraassay coefficient of variation of 17% and a high degree of accuracy. The concentration of LTB4 in serum collected with (NDGA) was less than 10% of that observed in blood collected without the lipoxygenase inhibitor. Ex vivo formation can therefore be a major obstacle in assessing circulating levels of LTB4.     

Structural elucidation of in vivo metabolites of penehyclidine in rats by the method of liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and isotope ion cluster

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography-electrospray ionization mass spectrometry, gas chromatography-mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS(n) spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.     

Delayed development of hypertension and increased aortic relaxation in spontaneously hypertensive rats after neonatal sympathectomy

The effect of neonatal sympathectomy on vasodilator responses to acetylcholine (ACh) and cAMP has been studied in aortic rings of spontaneously hypertensive rats (SHR) and normotensive animals. The relaxation of intact SHR aorta in response to ACh and cAMP was 20-35% lower than that of normotensive rats. Sympathectomy in normotensive rats did not affect the level of blood pressure and aorta reactivity to Ach. In SHR, sympathectomy caused a decrease in blood pressure, while relaxation in response to ACh and cAMP increased, as compared to intact SHR, but remained lower than in normotensive rats. The data obtained suggest that the decrease in arterial pressure of sympathectomized SHR is a result not only of the reduction in sympathetic effects but also of the increase in smooth muscle relaxation.     

Changes in the content of atrial natriuretic factor with the progression of hypertension in spontaneously hypertensive rats

In order to assess possible roles of atrial natriuretic factor (ANF) in spontaneously hypertensive rats (SHR), we examined the content of immunoreactive-ANF in plasma, the atria, hypothalamus and pons of SHR and Wister Kyoto (WKY) rats by radioimmunoassay at different stages of age. With the progression of hypertension, plasma concentration of ANF increased whereas it decreased in the atria in SHR. This suggests ANF is secreted in response to hypertension. On the other hand, at hypothalamus and pons, ANF content was significantly higher in SHR than in WKY rats. This finding suggests possible involvement of ANF in the central regulation of blood pressure.     

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining cyanide And its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 μmol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 μmol/ml for cyanide and from 0.01 to 1.0 μmol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using this method, the blood levels of two victims who had died from cyanide poisoning were determined.     

Determination of copper in urine and serum by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using enriched 65Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N'-ethylenebis-(trifluoroacetylacetoneimine) [H2(enTFA2)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H2(enTFA2) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a 10-ng level using H2(enTFA2). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H2(enTFA2) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data.     

Analysis of testosterone and dehydroepiandrosterone in saliva by gas chromatography-mass spectrometry

Testosterone and 3 beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) have been identified in human parotid fluid and saliva by gas chromatography-mass spectrometry/selected ion monitoring analyses of the t-butyldimethylsilyl ether and methyl oxime, t-butyldimethylsilyl ether derivatives. High specificity of analysis has been achieved by the use of high mass spectrometric resolution or by the monitoring of metastable peaks. Quantitative analyses indicate concentrations of both unconjugated testosterone and unconjugated dehydroepiandrosterone in the range 200-800 pmol/l in the saliva and parotid fluid of the normal males examined. These represent 1.5-7.5% of the concentrations of the steroids in blood plasma taken from the same subjects.     

Dynamic characteristics of baroreflex neural and peripheral arcs are preserved in spontaneously hypertensive rats

Although baroreceptors are known to reset to operate in a higher pressure range in spontaneously hypertensive rats (SHR), the total profile of dynamic arterial pressure (AP) regulation remains to be clarified. We estimated open-loop transfer functions of the carotid sinus baroreflex in SHR and Wistar Kyoto (WKY) rats. Mean input pressures were set at 120 (WKY??? and SHR???) and 160 mmHg (SHR???). The neural arc transfer function from carotid sinus pressure to efferent splanchnic sympathetic nerve activity (SNA) revealed derivative characteristics in both WKY and SHR. The slope of dynamic gain (in decibels per decade) between 0.1 and 1 Hz was not different between WKY??? (10.1 ± 1.0) and SHR??? (10.4 ± 1.1) but was significantly greater in SHR??? (13.2 ± 0.8, P < 0.05 with Bonferroni correction) than in SHR???. The peripheral arc transfer function from SNA to AP showed low-pass characteristics. The slope of dynamic gain (in decibels per decade) did not differ between WKY??? (-34.0 ± 1.2) and SHR??? (-31.4 ± 1.0) or between SHR??? and SHR??? (-32.8 ± 1.3). The total baroreflex showed low-pass characteristics and the dynamic gain at 0.01 Hz did not differ between WKY??? (0.91 ± 0.08) and SHR??? (0.84 ± 0.13) or between SHR??? and SHR??? (0.83 ± 0.11). In both WKY and SHR, the declining slope of dynamic gain was significantly gentler for the total baroreflex than for the peripheral arc, suggesting improved dynamic AP response in the total baroreflex. In conclusion, the dynamic characteristics of AP regulation by the carotid sinus baroreflex were well preserved in SHR despite significantly higher mean AP.     

Increased vascular contractile sensitivity to serotonin in spontaneously hypertensive rats is linked with increased turnover of phosphoinositide

This study was conducted to determine if increased vascular contractile sensitivity to serotonin in spontaneously hypertensive (SHR) rats is linked with increased phosphoinositide turnover. Aortic and mesenteric artery rings from SHR exhibited 6.2- and 5.0-fold greater contractile sensitivity to serotonin than the aortic and mesenteric artery rings from normotensive Wistar-Kyoto (WKY) rats. Serotonin-induced turnover of phosphoinositide was measured by quantifying the accumulation of [3H] inositol labeled inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3). Serotonin (3, 30, 200 microM) induced significantly greater accumulation of IP in SHR (279%, 590%, 895%) than in WKY (24%, 127%, 328%) aortic rings. Similarly, 3, 30 and 200 microM serotonin induced significantly greater accumulation of IP2 (118%, 241%, 451%) and IP3 (90%, 100%, 247%) in SHR than the accumulation of IP2 (15%, 58%, 122%) and IP3 (19%, 27%, 73%) in WKY aortic rings. Based on these data it is suggested that the greater vascular sensitivity to serotonin in SHR, at least in part, is attributable to increased turnover of phosphoinositide.     

Cardiac mitochondrial function and tissue remodelling are improved by a non-antihypertensive dose of enalapril in spontaneously hypertensive rats

Renal and cardiac benefits of renin-angiotensin system inhibition exceed blood pressure (BP) reduction and seem to involve mitochondrial function. It has been shown that RAS inhibition prevented mitochondrial dysfunction in spontaneously hypertensive rats (SHR) kidneys. Here, it is investigated whether a non-antihypertensive enalapril dose protects cardiac tissue and mitochondria function. Three-month-old SHR received water containing enalapril (10 mg/kg/day, SHR+Enal) or no additions (SHR-C) for 5 months. Wistar-Kyoto rats (WKY) were normotensive controls. At month 5, BP was similar in SHR+Enal and SHR-C. In SHR+Enal and WKY, heart weight and myocardial fibrosis were lower than in SHR-C. Matrix metalloprotease-2 activity was lower in SHR+Enal with respect to SHR-C and WKY. In SHR+Enal and WKY, NADH/cytochrome c oxidoreductase activity, eNOS protein and activity and mtNOS activity were higher and Mn-SOD activity was lower than in SHR-C. In summary, enalapril at a non-antihypertensive dose prevented cardiac hypertrophy and modifies parameters of cardiac mitochondrial dysfunction in SHR.     

Desaturase activities are depleted before and after weaning in liver microsomes of spontaneously hypertensive rats

In the present study, we have investigated the microsomal linoleic acid desaturation steps into arachidonic acid in 10- and 30-day-old spontaneously hypertensive rats (SHR), as compared to their normotensive control rats, Wistar Kyoto (WKY). Suckled by adoptive Wistar normotensive female, the SHR and WKY were fed the same diet. Our results show lower Delta 6 and Delta 5 desaturase activities (the limiting steps in the bioconversion of linoleic acid into arachidonic acid) in the young SHR, as compared to the WKY normotensive rats. The fatty acid composition of liver microsomal total lipids evidences a higher proportion of linoleic acid in SHR than in WKY, in agreement with the partially depleted desaturase activities. Such a loss of desaturase activities may be under the control of hormones involved in the regulation of SHR blood pressure.     

Bile acids are able to reduce blood pressure by attenuating the vascular reactivity in spontaneously hypertensive rats

Effects of the synthetic bile acids on blood pressure were examined in spontaneously hypertensive rats. Continuous intravenous administration of the bile acids at the rate of 1 mg/min for 20 min significantly lowered the blood pressure by 12 mmHg. In order to examine its blood pressure lowering mechanism, the isolated mesenteric arterial perfusion system was employed. Bile acids in the perfusate inhibited vascular reactivity to norepinephrine and KCl in a dose-dependent manner. This inhibitory action diminished as the concentration of potassium in the perfusate decreased. When the perfusate was free from potassium, its inhibitory action completely disappeared. These results in vivo and in vitro studies strongly suggest that bile acids act directly on the vascular beds and attenuate vascular response to norepinephrine.     

Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10⁶ DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10⁶ DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Occurrence of 4-hydroxyalkenals in rat tissues determined as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry

Malondialdehyde measurements have been the major tool for studying relationships between lipid peroxidation and tissue pathology. Recently, we presented a novel gas chromatography-mass spectrometry method for direct detection of phospholipid peroxides with picogram sensitivity based on transesterification of phospholipids or triglycerides to form pentafluorobenzyl esters. Under some circumstances the reactive primary oxidation products break down. Therefore, we developed a convenient, high sensitivity method to detect more stable secondary lipid oxidation products, the 4-hydroxyalkenals. The method accomplishes a facile extraction of 4-hydroxynonenal from tissues by forming pentafluorobenzyl oxime derivatives to displace aldehydes from Schiff base linkages. 4-hydroxynonenal was found in heart, liver, adrenal, and testis from rats and was detected to the 10-100 pg level by the current method.     

Detection of microbial-derived fatty acids in carious dentin by gas chromatography and gas chromatography-mass spectrometry

The aim of the present study was to analyze the fatty acid content of carious and sound human dentin. Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C₁₀-C₁₈ size in the carious dentin, whereas fatty acids of C₁₆ size were present in minute amounts in three samples of the corresponding sound dentine controls. No fatty acids were detected in the other sound dentin control samples. The source of fatty acids was considered to be microorganisms invading the dentin during the progression of the caries lesion. The presence of bacterial fatty acids in carious dentin may serve as a marker for the pathological process and thus contribute to the understanding of the mechanisms involved.