Determination of D-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography

A column-switching chiral HPLC system for the determination of minute amounts of D-Ala in mammalian tissues has been established. D-Ala and its L-enantiomer are purified as a DL mixture on a micro-ODS column after precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and are introduced to a chiral column to determine each enantiomer. The calibration curve of D-Ala spiked into a rat cerebellum sample is linear from 5 to 5000 fmol with a correlation coefficient of 1.0000. The lower limit of quantitation of D-Ala is 5 fmol (S/N=5). Within-day and day-to-day precisions of spiked D-Ala (15 fmol) are 3.9 and 4.8% (R.S.D), respectively. With this system, the anatomical distribution of free D-Ala in the rat central nervous system and periphery has been investigated. Among the 22 examined tissues of the rat, the highest amount of D-Ala has been observed in the anterior pituitary gland (86.4+/-9.9 nmol/g wet tissue), and the second highest amount has been observed in the pancreas (29.2+/-5.0 nmol/g wet tissue). Postnatal and day-night changes in D-Ala amounts in the anterior pituitary gland have also been studied. The amount of D-Ala is highest at 6 weeks of age and significantly decreases with age, and the amount of D-Ala is significantly higher during the daytime than during the nighttime.     

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues

A simple and selective method for the determination of anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand, and its analogs with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) was developed. The calibration curve for standard anandamide was linear over the range 625 fmol-125 pmol per injection (r=0.999) with a precision of 1.0% (C.V.) at 25 pmol. The detection limit attained was 200 fmol per injection at a signal-to-noise ration of 2. Anandamide and its analogs were extracted from rat brain and peripheral tissues according to the method of Folch, and the recovery of anandamide from rat brain homogenates was 67.0–72.6%. The method was applied to their determination in rat brain and peripheral tissues.     

Syntheses and biological activities of sandostatin analogs containing stereochemical changes in positions 6 or 8

In a continuation of our research efforts on the design and synthesis of novel peptidomimetic structures, we have synthesized a series of sandostatin amide analogs in which stereoisomers of threonine and beta-hydroxyvaline(beta-Hyv) are employed. The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) explore the effects on biological activity of stereochemical modifications and beta-methylation at positions 6 or 8. By these modifications, we examine the role of the two residues in binding to somatostatin receptors. We describe the synthesis and biological activity of these analogs. In combination with the results of the conformational analysis, this study provides new insights into the structural requirements for the binding affinity of somatostatin amide analogs to somatostatin receptors [Mattern et al., Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8].     

Quantification of malonyl-coenzyme A in tissue specimens by high-performance liquid chromatography/mass spectrometry

We present a validated high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the quantification of malonyl-coenzyme A (CoA) in tissues. The assay consists of extraction of malonyl-CoA from tissue using 10% trichloroacetic acid, isolation using a reversed-phase solid-phase extraction column, HPLC separation, and detection using electrospray MS. Quantification was performed using an internal standard ([(13)C(3)]malonyl-CoA) and multiple-point standard curves from 50 to 1000pmol. The procedure was validated by performing recovery, accuracy, and precision studies. Recoveries of malonyl-CoA were determined to be 28.8+/-0.9, 48.5+/-1.8, and 44.7+/-4.4% (averages+/-SD, n=5) for liver, heart, and skeletal muscle, respectively. Accuracy was demonstrated by the addition of known amounts of malonyl-CoA to tissue samples. The malonyl-CoA detected was compared with the malonyl-CoA added, and the resulting relationships were linear with slopes and regression coefficients equal to 1. Precision was demonstrated by repetitive analysis of identical samples. These showed a within-run variation between 5 and 11%, and the interbatch repeatability was essentially the same. This procedure was then applied to rat liver, heart, and skeletal muscle, where the malonyl-CoA contents were found to be 1.9+/-0.6, 1.3+/-0.4, and 0.7+/-0.2nmol/g wet weight, respectively, for these tissues. This analytical approach can be extended to the quantification of other acyl-CoA species with no significant modification.     

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues

Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75+/-5%. The detection limit was 10pmol UCB/g wet tissue. Variance was +/-2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (相似文献   

The myoglobin content in red, intermediate and white fibres of the swimming muscle sin three species of shark–a comparative study using high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure is described for the determination of myoglobin in extracts of small samples of tissue from the three different fibre types in the swimming muscles of three species of sharks, Etmopterus spinax, Galeus melastomus and Scyliorhinus canicula . The method, which is based on the separation of myoglobin from haemoglobin from haemoglobin based on HPLC using a gel permeation chromatography column, has a detection limit of about 3 pmol myoglobin (Mb). In addition it has the added advantage of specific identification by its Soret band absorption and quantification. In all three species, the three fibre types of the muscle are completely separated and can be isolated at a high degree of purity. In red fibres the myoglobin content varied between 565 nmol mg⁻¹ wet weight ( Scyliorhinus ) and 170 nmol mg⁻¹ wet weight ( Galeus ). Intermediate fibres contained from 215 to 57, and white fibres from 11 to zero nmol mg⁻¹ wet weight. The myoglobin content is closely correlated to the vascularization as well as to the amounts of mitochondria in the different fibre types.     

Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.     

Substance-P-related and neurokinin-A-related peptides from the brain of the cod and trout.

An extract of the brain of the rainbow trout, Oncorhynchus mykiss contained high concentrations of both neurokinin A-like immunoreactivity (corresponding to 90 pmol mammalian neurokinin A/g wet tissue) and substance-P-like immunoreactivity (corresponding to 50 pmol mammalian substance P/g wet tissue) measured by radioimmunoassay using antisera directed against the C-terminal regions of the mammalian peptides. In contrast, an extract of the Atlantic cod. Gadus morhua contained only neurokinin-A-like immunoreactivity (151 pmol/g). This apparent paradox was resolved by determination of the primary structures of the fish tachykinins. Trout substance P (Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-MetNH2) has the same amino acid sequence in its C-terminal region as that in the corresponding region of mammalian substance P. Cod substance P (Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-MetNH2), however, contains a substitution at position 8 (Phe----Ile) that abolishes reactivity with the antiserum to substance P but permits reactivity with the antiserum to neurokinin A. The amino acid sequence of cod and trout neurokinin A is the same (His-Lys-Ile-Asn-Ser-Phe-Val-Gly-Leu-MetNH2) and shows two substitutions (Thr3----Ile and Asp4----Asn) compared with mammalian neurokinin A. The data indicate that nervous tissue of teleost fish contain tachykinins that are analogous to the peptides found in mammalian tissues.     

High-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography of glycosphingolipids

A method combining high-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography is introduced for determination of glycosphingolipids. Glycolipids in crude extract from rat liver were separated quantitatively from neutral lipids and phospholipids with a phenylboronic acid-derivatized silica gel column. Glycolipids were eluted quantitatively with approximately 98% of crude extract recovered. This column is useful for selective cleanup of glycosphingolipids in crude extract from tissue. Simultaneously, a fluorometric determination of glycosphingolipids with 7-amino-4-methylcoumarin after NaIO4 oxidation on a TLC plate was introduced and its condition was optimized. Glycolipids in amounts ranging from 1 to 100 pmol are easily detectable and give linear responses over the respective ranges. The method is fast and useful for the determination of glycolipids from small amounts of biological samples and requires a minimum amount of about 1 mg of biological specimen for determination of glycolipids.     

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.     

A sensitive method for the quantitation of lysophosphatidylcholine in canine heart

We have developed a procedure for the determination of small amounts of lysophosphatidylcholine in cardiac tissue. Lysophosphatidylcholine from canine heart was separated from the major phospholipids by column chromatography, and then acetylated with labeled acetic anhydride. The acetylated lysophosphatidylcholine was isolated by thin-layer chromatography and the lysophosphatidylcholine content was calculated from the radioactivity associated with the acetylated product. Although the sensitivity of the assay depends on the specific radioactivity of the acetic anhydride used, as low as 0.5 nmol of lysophospholipid in tissue samples can be readily quantitated. The results obtained from the control and ischemic canine cardiac tissues by this assay compares favorably with those obtained by lipid-phosphorus assay. The sensitivity and specificity of the present procedure allows us and other investigators to assay for lysophosphatidylcholine content in very small (10 mg wet weight) tissue samples.     

N-Acetylsuccinimidylglutamate, a Cyclic Imide Form of the Peptide N-Acetylaspartylglutamate, Is Present in Low Micromolar Concentrations in Murine and Bovine CNS

Abstract: N -Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N -acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the α- and β-carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 ± 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 ± 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amounts in the superior colliculus and lateral genicutale nucleus (1.17 ± 0.05 and 0.82 ± 0.13 pmol/mg wet weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions ( r = 0.74, p < 0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)-NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.     

Simultaneous determination of psychosine and cerebrosides

A new method was developed for the simultaneous determination of psychosine and cerebrosides in tissues. Total lipids extracted from the tissues were treated with [3H]acetic anhydride in toluene-methanol. Known amounts of nonradioactive N-acetylpsychosine were added to the reaction product and then subjected to mild alkaline methanolysis. After the product was washed, it was fractionated by silica gel column chromatography and the fraction containing glycolipids was benzoylated. The benzoylated product was finally fractionated on TLC. The amounts of benzoylated derivatives of nonhydroxy- and hydroxycerebrosides and N-acetylpsychosine were determined using a scanning densitometer. The amounts of psychosine in tissues were calculated from the radioactivity in the spot of N-acetylpsychosine and the recovery of added carrier N-acetylpsychosine. This method allowed us to determine 5 to 1000 pmol of psychosine and 1 to 20 nmol of cerebrosides in peripheral nerves and other tissues of the twitcher mouse as well as transfected Schwann cells derived from the sciatic nerves.     

Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (～1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (～6.8pmol/g fresh tissue).     

Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino derivatives by high-performance liquid chromatography

A method for the quantitation of picomole amounts of neutral and amino sugars in glycoconjugates was developed. Glycoconjugates were hydrolyzed with a mixture of equal amounts of 4 M trifluoroacetic acid and 4 M hydrochloric acid, and the free amino groups were acetylated. Sugars were coupled with 2-aminopyridine. After the excess reagents were removed by gel-permeation high-performance liquid chromatography, the fluorescent pyridylamino derivatives of sugars were separated and quantified by high-performance liquid chromatography on a reversed-phase column. This method allowed the determination of 0.01-10 nmol of sugars. About 100 pmol of several glycoconjugates were analyzed by the present method, with satisfactory results.     

A rapid and safe method of estimating nanomole quantities of P, K+, Na+, Ca2+, and Mg2+ in plant material by perchloric acid digestion

A method for the determination of nanomole amounts of P and major cations in samples of dried plant tissue is described. Samples weighing 2-7 mg were digested by refluxing tissue in a mixture of perchloric and nitric acids, under conditions which minimized the hazards associated with perchloric acid digestion. In most tissues analyzed, reproducibility between triplicate samples was less than +/- 5%. However, where the amount of P in the tissue was very low (samples contained about 50 nmol P each), the variation between triplicates was greater.     

Estimation of acyldihydroxyacetone phosphate and lysophosphatidate in animal tissues

Chemical and enzymatic methods have been developed to measure small quantities (10(-8) - 10(-10) mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl3/methanol were subjected to solvent partitioning at two different pH values for partial purification of this keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB3H4 or by enzymatic reduction with [4B-3H]NADPH using a partially purified acyldihydroxyacetone-phosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB3H4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2-3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxyacetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 +/- 0.64 (guinea pig liver), 4.3 +/- 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.     

Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at −80 °C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355 ± 0.030 and 0.827 ± 0.082 units of nmol/g wet tissues in brain, 0.320 ± 0.019 and 0.293 ± 0.035 units of nmol/g wet tissues in kidney, 0.326 ± 0.064 and 0.213 ± 0.029 units of nmol/g wet tissues in liver, and 0.364 ± 0.015 and 0.349 ± 0.023 units of nmol/g wet tissues in heart (means ± SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.