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PubMed1998

Sequencing using capillary electrophoresis of short tandem repeat alleles separated and purified by high performance liquid chromatography.

Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene-divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion-pairing reverse-phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double-stranded DNA sequencing properties. In this study, the IP-RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/ FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak-captured, then cycle-sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 microm and a total length of 47 centimeters. The total analysis time including capillary filling and pre-electrophoresis was less than 30 min for a 220-bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9, 9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.

Alleles↗

PubMed2003

Characterization of pharmaceutical drugs by a modified nonaqueous capillary electrophoresis--mass spectrometry method.

A simple method for the separation and characterization of a group of nine basic compounds, comprising seven tricyclic antidepressant and two bronchodilator drugs, by nonaqueous capillary electrophoresis (NACE) employing ultraviolet and mass spectrometry detection is described. After optimization of the electrophoresis separation conditions, including the compositions of the electrolyte and the organic solvent, a reliable separation of all nine basic analytes was achieved in 80 mM ammonium formate dissolved in a methanol-acetonitrite (80:20 v/v) mixture, having an apparent pH of 8.7. The volatile nonaqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by mass spectrometry. When results were compared with reversed-phase gradient and isocratic high-performance liquid chromatography (HPLC) methods, the NACE method provided greater efficiency, achieving baseline resolution for all nine basic compounds in less than 30 min. The NACE method is suitable for use as a routine procedure for the rapid separation and characterization of basic compounds and is a viable alternative to HPLC for the separation of a wide range of pharmaceutical drugs.

Antidepressive Agents, Tricyclic↗

PubMed2005

Validated HPLC assay for iron determination in biological matrices based on ferrioxamine formation.

A simple, robust and reproducible HPLC method has been developed and validated for iron determination in biological matrices. It is based on chelation with desferrioxamine (DFO) and the measurement of the chelate ferrioxamine (FO). The method was developed to permit monitoring of iron bio-kinetics and estimation of iron status in experimental animals. The chromatography was performed on a stainless steel XTerra MS C18 column (Waters; 250 mm x 4.6 mm i.d., 5 microm) using a gradient of Tris-HCl buffer (10mM, pH 5) and acetonitrile. The method was validated in terms of selectivity, linearity (0.3-80 nmol on-column), limit of detection (0.2 nmol on-column), low limit of quantification (0.3 nmol on-column), recovery (91-102%), intra- and inter-day reproducibility, stability, and robustness. The method's universal applicability was illustrated by monitoring plasma and heart iron kinetic profiles in rats after a single intraperitoneal (i.p.) injection of 200mg/kg iron dextran.

Animals↗

PubMed1995

Separation of hemoglobin acetaldehyde adducts by high-performance liquid chromatography-cation-exchange chromatography.

A HPLC-based method was developed to provide a simple way to study changes to hemoglobin induced by acetaldehyde in vitro. This method distinguished 18 human hemoglobin fractions including a new acetaldehyde-induced fraction HbA1ach3. The method consists of a Poly CAT A cation-exchange column and a stepwise salt and pH gradient, with a total analysis time of 31 min. The formation of acetaldehyde adducts was studied by incubation of hemoglobin with different Ach concentrations (5-1000 microM) and different incubation times (0-48 h). Physiological (5-250 microM) Ach concentrations induced increases mainly in 3 known fractions: HbA1ach1, HbA1prec, and HbA1d3; plus, it caused the formation of a new fraction, HbA1ach3. The specificity of the changes to acetaldehyde was studied by incubation of hemoglobin with glucose and acetylsalicylic acid. HbA1ach3 was the only acetaldehyde-induced hemoglobin fraction which was not also increased by glucose and acetylsalicylic acid treatment. The formation of HbA1ach3 showed a dose and time dependence on acetaldehyde incubations. Dialyzation and reduction experiments showed that HbA1ach3 is a stable adduct of hemoglobin, and incubation with purified HbAO showed that HbA1ach3 is an adduct of HbAO. The within-run and between-run coefficients of variation for HbA1ach3 (0.83% of total hemoglobin) were 10.8 and 15.1%, respectively, and the analytical recovery was 82-97%. These results indicate that in addition to the new, acetaldehyde-specific fraction HbA1ach3, several other types of hemoglobin adducts were formed with acetaldehyde. The current method might be useful in clarifying the relationships between hemoglobin and acetaldehyde in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗

PubMed1984

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography.

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.

Animals↗

PubMed1997

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

Affinity Labels↗

PubMed1991

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.

6-Ketoprostaglandin F1 alpha↗

PubMed1986

An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling.

A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.

Animals↗

PubMed2005

Determination of quinolones and fluoroquinolones in hospital sewage water by off-line and on-line solid-phase extraction procedures coupled to HPLC-UV.

In this work, the development of two solid-phase extraction procedures (off-line and on-line formats) for the identification and quantification of several (fluoro)quinolones in hospital sewage water by HPLC-UV is described. Both procedures are based on the use of C18 and anion exchange (SAX) sorbents for the preconcentration and clean-up steps, respectively, and all variables influencing both steps were optimised. In the off-line format, after its pH was adjusted to 2.5, sample was preconcentrated on a C18 cartridge and eluted with 4 mL of methanol/ammonia (94/6). The methanolic extract must be diluted up to 10 mL with water to allow quantitative retention of the analytes on the SAX cartridge. In the on-line format, the addition of 2.5% of NH4Cl to the sewage water sample (pH = 2.5) was necessary to increase the breakthrough volumes of the analytes in the C18 precolumn. Quantitative transfer of the (fluoro)quinolones from the C18 precolumn to the SAX precolumn was accomplished by pumping 2 mL of a mixture methanol/water (40/60, pH = 9.2) at 2 mL min(-1). Elution of the analytes from the SAX precolumn by means of the chromatographic mobile phase required the inclusion of an additional isocratic step at the beginning of the gradient program. Both off-line and on-line solid phase extraction procedures coupled to HPLC-UV were applied to the analysis of a sewage water sample collected in the sewer system at the output of the St Dimphna Hospital (Geel, Belgium). The fluoroquinolone ciprofloxacin was found in this sample and quantified at 5.8 +/- 0.4 microg L(-1) (off-line method) and 5.6 +/- 0.5 microg L(-1) (on-line method). The analysis of spiked samples containing the seven (fluoro)quinolones studied provided quantitative recoveries in all cases with low RSD values (from 6 to 12%), and all the analytes could be identified by means of their UV spectra with match factors varying from 950 to 985 depending on the (fluoro)quinolone.

Chromatography, High Pressure Liquid↗

PubMed2006

HPLC analysis of functionalized poly(amidoamine) dendrimers and the interaction between a folate-dendrimer conjugate and folate binding protein.

Poly(amidoamine) (PAMAM) dendrimers of different generations with carboxyl, acetyl, and hydroxyl terminal groups and a folic acid (FA)-dendrimer conjugate were separated and analyzed using reverse-phase high performance liquid chromatography (HPLC). Analysis of both the individual PAMAM derivatives and the separation of mixed generations can be achieved using a linear gradient 0-50% acetonitrile (ACN) (balance water) within 40 min. We also show that PAMAMs with defined acetylation and carboxylation degrees can be analyzed using HPLC. Furthermore, a generation 5 dendrimer-FA conjugate (G5.75Ac-FA4; Ac denotes acetyl) was analyzed and its specific binding with a bovine folic acid binding protein (FBP) was monitored. The HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicate the formation of three complexes after the binding of G5.75Ac-FA4 with FBP. Dendrimers with FA moieties show much higher specific binding capability with FBP than those without FA moieties. Findings from this study indicate that HPLC is an effective technique not only for characterization and separation of functionalized PAMAM dendrimers and conjugates but also for investigation of the interaction between dendrimers and biomolecules.

Biocompatible Materials↗

PubMed2001

DNA melting analysis for detection of single nucleotide polymorphisms.

BACKGROUND: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. METHODS: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). RESULTS: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15-167 bp in length and differing by only a single nucleotide substitution. CONCLUSIONS: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.

Base Pair Mismatch↗

PubMed1993

Determination of oxiracetam in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection.

Reversed-phase HPLC methodology utilizing pre-column derivatization and post-column reaction fluorimetric detection has been developed and applied to the determination of oxiracetam in human plasma. The method involves preliminary isolation of oxiracetam and internal standard from plasma by solid-phase extraction prior to the formation of their n-propyl carbamate derivatives. The carbamate derivatives were subsequently isolated by solid-phase extraction and subjected to a gradient liquid chromatographic separation on an octadecylsilica column prior to on-line post-column alkaline hydrolysis to produce the corresponding primary amine, which was in turn derivatized with o-phthalaldehyde and 3-mercaptopropionic acid to yield a fluorescent isoindole. The isoindole was then quantified using a fluorescence detector. The method provided an on-column detection limit of 0.5 ng of oxiracetam and was sufficiently sensitive, accurate, and precise to support pre-clinical or clinical pharmacokinetic studies.

Chromatography, High Pressure Liquid↗

PubMed1993

Two-dimensional high-performance liquid chromatographic method for assaying S-adenosyl-L-methionine and its related metabolites in tissues.

S-Adenosyl-L-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decarboxylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0-10% acetonitrile gradient in 10 mM ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 microns, 250 mm x 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15-400 mM), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30-500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9-22% for rat liver and 6-24% for mast cells.

Animals↗

PubMed1998

Determination of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection.

A new direct HPLC method with fluorescence detection has been developed for the routine analysis of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide, in wines and other beverages. These compounds are the main agents responsible for the "taste of light" that some white wines and other beverages develop when they are exposed to the light, due to the formation of sulfur compounds that produce an anion/garlic odor. A Hewlett-Packard 1100 gradient liquid chromatograph with 1046A fluorescence detector was used. To improve the selectivity, each compound was monitored to fit the best lambda excitation/lambda emission (265/525 nm). A 500 nm cut-off filter was used. The column was a Hypersil C18 ODS, 200 x 2.1 mm, 5 microns particle size. The volume injected was 20 microliters. A constant flow-rate of 0.6 ml/min was used with two solvents: solvent A, 0.05 M buffer NaH2PO4 at pH = 3.0 with H3PO4 and solvent B, acetonitrile. The precision, linearity and sensitivity of this method have been established.

Beverages↗

PubMed1993

A human epithelial cell line, intestine 407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B4.

The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 microM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-14C]arachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB4 were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB4. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed.

Calcimycin↗

PubMed1984

Corticosteroid metabolism in isolated rat kidney in vitro. I. Formation of lipid soluble metabolites from corticosterone (B) in renal tissue from male rats.

Kidneys of male Sprague Dawley rats have been isolated and perfused in vitro in order to study the metabolism of corticosterone (B). B is the main endogenous corticosteroid in this species. Using 3H-B and HPLC for the separation of steroid metabolites it has been possible to detect radioactive derivatives of B which have been denoted as met I, II and III. These substances were purified and compared with authentic reference hormones under different isocratic and gradient elution techniques. We observed chromatographic identity of met I with 11-dehydro-20-dihydro-B, of met II with 20-dihydro-B and of met III with 5 alpha-H-4,5-dihydro-B. From the fact that conversion of B can be observed with normal (50 g X l-1 albumin in perfusate) and elevated (75 g X l-1) colloid osmotic pressure of the recirculating perfusate it can be concluded that B gets access to the metabolic site in renal tissue not solely by glomerular filtration and tubular reabsorption. The metabolites identified presently are excreted in the urine. Metopirone increased the concentration of met I and decreased the concentration of met II. This is compatible with the concept of a stimulatory effect of metopirone on a C-20-hydroxysteroid oxidoreductase and a C-11-hydroxysteroid dehydrogenase.

Animals↗

PubMed1996

Mature human atherosclerotic plaque contains peroxidized phosphatidylcholine as a major lipid peroxide.

The initial stage of atherosclerotic plaque formation involves oxidation of the phosphatidyl-choline moiety of low density lipoprotein (LDL) and subsequent uptake by macrophages. Ongoing uptake in developing plaque also may involve oxidized LDL and would require an oxidizing environment in plaque lipids. Atherosclerotic plaque lipids from 12 patients undergoing peripheral vascular procedures were extracted in chloroform: methanol (2:1). This extract was applied to a 25 cm 5 micron silica HPLC column and eluted with a ternary gradient mobile phase utilizing a laser light scattering (ELSD) mass detector. Individual lipid fractions were then analyzed. Cholesterol, both free and esterified, was the most prominent lipid in plaque (104 +/- 74 mg/gm tissue. However, lipid peroxides were present in much higher concentrations (3.52 +/- 2.84 FU X 10(4)/mg phospholipid) and overall level (21.27 +/- 10.10 FU X 10(4)/gm plaque) in the phospholipid component (*p< 0.05). Phosphatidyl-choline (PC) accounted for 63% of the total phospholipid peroxides recovered (6.31 +/- 5.09 mg/gm plaque; *p<0.05). PC and phosphatidylinositol (PI) content were linearly related to lipid peroxide fluorescence (PC; r=0.696; p=0.01) (PI; r=0.809; p=0.001). Lipid peroxides in human atherosclerotic plaque are present primarily in the phospholipid component and phosphatidyl-choline forms the bulk of these peroxides. PC may play an important role in ongoing plaque lipid accumulation.

Analysis of Variance↗

PubMed1995

Analysis of lipoxygenase kinetics by high-performance liquid chromatography with a polymer column.

Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio- and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX-catalyzed oxidation of mixtures of lipids containing linoleate, using high-performance liquid chromatography (HPLC) and a light-scattering detector (LSD). Previous literature suggested that reversed-phase HPLC with silica-based columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed-phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one-tenth the rate of linoleic acid. The diacylglycerol, 1,3-dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono- and dihydroperoxides as well as other unidentified products.

Chromatography, High Pressure Liquid↗