Preparation of lipid-free tissue extracts for chromatographic determination of thyroid hormones and metabolites

We have developed a new method of preparing tissues for analysis of thyroid hormones and metabolites which eliminates troublesome lipids and proteins. Frozen tissue is homogenized by grinding with dry ice in a Waring blender, and the moist powder obtained is extracted with chloroform:methanol (2:1). In a modification of the Folch procedure for the total separation of lipids, the filtered extracts then are partitioned into polar and nonpolar layers by the addition of 0.05% aqueous calcium chloride. The upper phase contains the iodocompounds of interest as well as all salts and small polar molecules. The lipids remain dissolved in the lower phase after it is back-extracted with pure upper phase. The combined upper or aqueous methanol layers are lyophilized and the residue is taken up in methanol to yield a concentrated solution ready for analysis of iodocompounds. The greater clarity of the extract permits application of larger samples for two-dimensional paper chromatography than has been customary. For gradient analysis by reverse-phase ion-pair high-performance liquid chromatography (HPLC), the nitrogen-evaporated methanol extract is dissolved in the initial mobile phase, 0.1% H₃PO₄, for injection onto the column. Using these methods we have achieved the first reported separation of ¹²⁵I-labeled tissue iodothyronine metabolites by HPLC. The new method of extraction is of general applicability to any biological sample which might be analyzed in thyroid hormone metabolism research.     

Characterization of neuropeptides by reversed-phase, ion-pair liquid chromatography with post-column detection by radioimmunoassay : Application to thyrotropin-releasing hormone, substance P, and vasopressin

Neuropeptide contents of rat brain samples were determined by radioimmunoassay (RIA) after fractionation of tissue extracts by high-performance liquid chromatography (HPLC). Solvent systems were composed of acetic acid, acetonitrile and short-chain (5–8 carbons) alkylsulfonic acids. Separate solvent systems were developed for thyrotropin-releasing hormone, substance P, arginine vasopressin and biologic analogs, and the enkephalins. All separation systems tested gave 80–90% recovery of picogram quantities of peptides. When lyophilized, the HPLC solvents did not interfere significantly with the RIAs, allowing quantitation of tissue concentrations of isolated neuropeptides using the lyophilized eluent from the HPLC. The combination of liquid chromatography with RIA should allow for very accurate identification and quantification of peptides in biologic samples containing large numbers of potentially cross-reacting species of molecules.     

Determination of the anticonvulsant felbamate and its three metabolites in brain and heart tissue of rats

An automated, internal standard high-performance liquid chromatographic method for the simultaneous quantitation of felbamate and its three metabolites in adult and neonatal rat brain and heart tissue homogenates was developed and validated. The homogenates prepared from one part of the tissue and four parts of water were extracted with ethyl acetate, and the extract was evaporated to dryness and redissolved in mobile phase. Separation was accomplished on a Waters Resolve C₁₈, 5 μm, 300 mm × 3.9 mm I.D. column with a mobile phase consisting of 0.01 M phosphate buffer, pH 6.8—acetonitrile—methanol (800:150:50, v/v/v). Eluting peaks were monitored with an ultraviolet detector at 210 nm. The linear range of the assay for felbamate and the metabolites was 0.20–50.00 μg/ml of homogenate or 1–250 μg/g of brain or heart tissue. The lower limit of quantitation for all four analytes was 0.20 μg/ml of homogenate or 1.00 μg/g of tissue.     

Detection of the toxin domoic acid from clam extracts using a portable surface plasmon resonance biosensor

The amnesic shellfish poison domoic acid is produced by marine algae of the genus Pseudo-nitzschia. We have developed a portable surface plasmon resonance (SPR) biosensor system for the detection of domoic acid. Because of concerns with domoic acid contamination of shellfish, there is a need for rapid field quantification of toxin levels in both shellfish and seawater. Antibodies were raised against domoic acid and affinity purified. These antibodies were used to develop competition- and displacement-based assays using a portable six-channel SPR system developed in our laboratories. Standard curves for detection of domoic acid in phosphate buffered saline and in diluted clam extracts analyzed by the competition-based SPR assay demonstrated a limit of detection of 3 ppb (10 nM) and a quantifiable range from 4 to 60 ppb (13–200 nM). Comparison of analyses for domoic acid levels in Pacific razor clams, Siliqua patula, containing moderate to high levels of domoic acid by the standard HPLC analysis protocol and the SPR-based assay gave an excellent correlation. This same technology should also function for detection of domoic acid in concentrated algal extracts or high dissolved levels in seawater.     

Strong Association of Unesterified [3H]Docosahexaenoic Acid and [3H-Docosahexaenoyl]Phosphatidate to Rhodopsin During In Vivo Labeling of Frog Retinal Rod Outer Segments

Docosahexaenoic acid (DHA, 22:6n-3), the most prevalent fatty acid in phospholipids of rod outer segments (ROS), is essential for visual transduction and daily renewal of ROS membranes. We investigated the association of [³H]DHA-lipids to rhodopsin in ROS from frogs (Rana pipiens) after in vitro (4 hrs) and in vivo (1 day and 32 days) labeling. Lipids from lyophilized ROS were sequentially extracted with hexane (neutral lipids), chloroform:methanol (phospholipids) and acidified chloroform:methanol (acidic phospholipids). After in vitro labeling, free [³H]DHA was easily extracted with hexane (66% of total ROS free DHA), implying a weak association with proteins (rhodopsin). In contrast, after in vivo labeling free [³H]DHA was mainly recovered in the acidic solvent extract (89–99%). Of all phospholipids, [³H-DHA]phosphatidic acid (PA) displayed the highest binding to rhodopsin after both in vitro (43% in acidic extract) and in vivo (>70%) labeling suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction.     

Simultaneous determination of dopamine, dopac and homovanillic acid : Direct injection of supernatants from brain tissue homogenates in a liquid chromatography—electrochemical detection system

A simple method based on high-performance liquid column chromatography with electrochemical detection is described for the simultaneous determination of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in discrete brain regions of rats. The supernatant of a tissue homogenate is injected directly onto a liquid chromatograph, thus omitting the commonly adopted adsorption step. Of the four different supports tested Nucleosil C₁₉ (5 μm) was found superior with respect to chromatographic performance. The effects of pH, methanol and the ion-pairing agent hexyl sulfate on the retention were studied. The mobile phase used in the final studies consisted of citrate buffer pH 4.25—methanol (92:8, v/v) containing hexyl sulfate (1.7 · 10⁻³M). Standard curves of dopamine, DOPAC and HVA were found linear up to about 600 pmol per injection for each compound. The precisions of the chromatographic step were (s_rel. %): 0.72% (dopamine), 1.26% (DOPAC) and 2.69% (HVA).     

Simultaneous quantitation of etoposide and its catechol metabolite in human plasma using high-performance liquid chromatography with electrochemical detection

Etoposide, a highly active and widely used antineoplastic agent, is O-demethylated to its active catechol metabolite. A high-performance liquid chromatographic assay method for the simultaneous quantitation of etoposide and etoposide catechol in human plasma was established. Etoposide and etoposide catechol were extracted from plasma using chloroform and methanol followed by phase separation, evaporation of the organic phase, and reconstitution of the residue. Chromatography was accomplished using a reversed-phase phenyl analytical column (390 mm×3.9 mm I.D.) with a mobile phase of 76.6% 25 mM citric acid–50 mM sodium phosphate (pH 2.4)–23.4% acetonitrile pumped isocratically at 1 ml/min with electrochemical detection. The limit of detection for etoposide was 1.2 nM and for etoposide catechol was 0.2 nM. The precision (CV) for etoposide ranged from 0.7 to 3% and for the catechol metabolite from 1 to 6%; accuracy of predicted values ranged from 97 to 106% and 94 to 103%, respectively. The assay was linear from 0.1 to 10 μM for etoposide and from 0.005 to 0.5 μM for etoposide catechol in plasma. Recovery of etoposide and etoposide catechol ranged from 93 to 95% and 90 to 98%, respectively. Stability of etoposide and etoposide catechol in human plasma containing ascorbic acid stored at −70°C for one year was demonstrated. This assay procedure is suitable for evaluation of etoposide and etoposide catechol pharmacokinetics in plasma following etoposide administration.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Human carbonic anhydrase isoenzyme C

Summary The effects of some alcohol and aldehyde containing fixatives on the antigenicity of human carbonic anhydrase isoenzyme C (HCA C) were tested in order to reveal the most suitable method for the immunohistochemical localization of this enzyme. The use of 2% and 4% paraformaldehyde or 2% glutaraldehyde solutions before immunoperoxidase (PAP) staining resulted in the loss of HCA C-specific reactivity in the surface epithelial cells of human appendicular and gastric mucosae, whereas the antigenic reactivity of HCA C was well retained in the parietal cells of gastric glands. In corresponding tissue sections fixed with one of the alcohol containing solutions (abs. methanol, methanol+chloroform 21 or Carnoy fluid) both the surface epithelial and parietal cells showed HCA C-specific immunostaining after the PAP procedure. In addition, the antigenicity of HCA C was found to be well preserved in some tubular cells of human kidney fixed in Carnoy fluid. The paraffin infiltration at relatively low temperature did not markedly affect the enzyme antigenicity. Fixation in Carnoy fluid coupled with paraffin embedding at 55–60° C in vacuo was found to give the best preservation of the antigenicity of HCA C with good morphological integrity for light microscopical localization.Grant support from the Finnish Culture Foundation     

Determination of fendiline and gallopamil by capillary isotachophoresis

Capillary isotachophoresis was applied for the determination of fendiline and gallopamil – calcium antagonists – in serum. The cationic electrolyte system containing Na⁺ with acetic acid as a counter constituent was used as a leading electrolyte with the pH 4.7 and the terminating electrolyte was β-alanine. Most of the proteins were precipitated with methanol, ethanol and dimethylketone. The lowest limits of quantitation were obtained for the pretreatment of serum with methanol. The recoveries of both compounds varied from 91.3 to 97.5%. The relative standard deviations varied from 0.6 to 7.7%.     

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C₈ preparative column with 6% acetonitrile—45% methanol—5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-μm Spin-X cartridges, applied to a reversed-phase C₁₈ column, and eluted in a 0–50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzylamiloride.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

The complete amino acid sequence of the antenna polypeptide B806-866-β from the cytoplasmic membrane of the green bacterium Chloroflexus auranliacus

The bacteriochlorophyll a-binding polypeptide B806–866-β was extracted from membranes of the green thermophilic bacterium Chloroflexus aurantiacus with chloroform/methanol/ammonium acetate. Purification of the antenna polypeptide (6.3 kDa) was achieved by chromatography on Sephadex LH-60, Whatman DE-32 and by FPLC. The complete amino acid sequence (53 amino acid residues) was determined. The B806–866-β polypeptide is sequence homologous to the antenna β-polypeptides of purple bacteria (27–40%) and exhibits the characteristic three domain structure of the B870, B800–850 and B800–820 antenna complexes. The two typical His residues, conserved in all antenna β-polypeptides of purple bacteria, were found: His-24 lies within the N-terminal hydrophilic domain and His-42 within the central hydrophobic domain. This polypeptide together with the previously described α-polypeptide form the basic structural unit of the B806–866 antenna complex from C. aurantiacus.     

Ganglioside sialidase activity in bovine neuronal perikarya

Neuronal perikarya were isolated, using bulk preparative procedures, from bovine brains. Synaptosomes, neuronal perikarya, and brain homogenates had similar ganglioside patterns, with the synaptosomes containing at least four times more total ganglioside per mg protein than the neuronal perikarya and twice that of the homogenate. Synaptosomes had 26–33 nmol total sialic acid/mg protein, while the neurons had only 15–17 nmol. Determination of ganglioside sialidase activity showed that neuronal perikarya had very low levels (negligible), in comparison with synaptosomes or whole-brain homogenates. Trypsin treatment during the isolation procedure enhanced sialidase activity two-to threefold in the particulate fraction of the whole-brain homogenate. Determination of the distribution of sialidase activity in the fractions obtained during the isolation of the neuronal perikarya showed that the sialidase activity was associated with the myelin, broken-off dendritic processes, and glial-cell fractions that banded in the less dense sucrose.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

High-performance liquid chromatography—thermospray mass spectrometry of epoxy polyunsaturated fatty acids and epoxyhydroxy polyunsaturated fatty acids from an incubation mixture of rat tissue homogenate

A method for the analysis of epoxy polyunsaturated fatty acids (EpPUFAs) and epoxyhydroxy polyunsaturated fatty acids (EpHPUFAs) in rat tissue homogenate, with homo-γ-linolenic acid (20:3, n − 6), arachidonic acid (20:4, n − 6), eicosapentaenoic acid (20:5, n − 3) or docosahexaenoic acid (22:6, n − 3) as a substrate, has been developed. Extraction with dichloromethane at pH 4–5 and concentration in the presence of pyridine were performed. Spectral analysis of chromatograms obtained with high-performance liquid chromatography—thermospray mass spectrometry showed the presence of EpPUFAs, EpHPUFAs and dihydroxy metabolites (DiHPUFAs) of EpPUFAs corresponding to each precursor fatty acid. On a selected-ion monitoring chromatogram, many EpPUFAs, EpHPUFAs and DiHPUFAs in an extract from an incubation mixture of each precursor fatty acid in aged rat tissue homogenate were detected simultaneously within 70 min. EpPUFAs and DiHPUFAs derived from 20:3 (n − 6) or 20:5 (n − 3) were detected in significant amounts. From these results, a highly active cytochrome P450 system or non-enzymic oxidative reactions in aged rat tissue homogenate were suggested.     

Nerve growth factor in skeletal tissues of the embryonic chick

Summary This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6–9 of incubation at 12 h intervals, and on days 10–18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24–36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.     

Suitability of the enzyme treatment and ammonium sulfate precipitation method for detection of staphylococcal enterotoxin from different foods

Summary An enzyme treatment and ammonium sulfate precipitation procedure was adapted to the detection of staphylococcal enterotoxin from high-protein foods. The enterotoxin is extracted from food with distilled water, after which soluble proteins are acid-precipitated (pH 4.5) and the supernatant washed with chloroform (pH 7.5). The extract is then treated with trypsin andPseudomonas peptidase for 2 h at +37°C. Residual unhydrolyzed material is precipitated with 60% ammonium sulfate for 15 min at +4°C. The precipitate is redissolved in phosphate buffer and concentrated by dialysis against polyethyleneglycol. The concentrate is washed with chloroform and lyophilized. The dry material is dissolved in 0.2 ml distilled water and enterotoxin detected by the micro-slide method with 24 h incubation at +37°C. Using this method, it has been possible within three days to detect 0.2–1.0 g staphylococcal enterotoxin A added to minced meat, dry sausage, smoked fish, cheese and milk.     

Improved Taxol production in suspension cultures of Taxus chinensis var. mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor

Taxus chinensis var. mairei was grown in two-liquid-phase culture in a 2.5 l airlift external loop reactor for production of Taxol. A mixture of oleic acid and dibutyl phthalate (1:1, v/v) in a volume ratio of 8:92 to the culture medium gave higher production of Taxol (up to 12 mg l^–1) with an aeration rate of 1 vvm. By combination of the in situ extraction, precursor feeding and additional sugar introduction, Taxol production (16.7 mg l^–1) was 5 times higher than that in the case without any additives (3.4 mg l^–1) and 1.4 times of that in the case with only thein situ extraction (12 mg l^–1). Taxol was extracted from the organic solvents with water and methanol (1:1:1.4, by vol.) and lyophilized giving a total yield of ca. 90%.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.