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H J Issaq

Publications and source records attributed to H J Issaq.

At least 37 records · Page 2Linked to original sources
PubMed1997

High-speed electrophoretic separation of DNA fragments using a short capillary.

Capillary electrophoresis using a replaceable gel buffer was applied to the separation of DNA fragments. A short effective length capillary (1-2 cm) at low electric field allowed the separation of a 20-1000 bp ladder in 1 min. Although similar separation speed was achieved with a longer capillary at high field, the resolution of larger fragments was degraded. The short effective length capillaries were able to separate the wildtype and mutant PCR products of the TGF-beta1 gene in under 45 s.

DNA↗
PubMed1997

The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products.

This work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2-7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.

Buffers↗
PubMed1997

Capillary electrophoresis of natural products.

Capillary electrophoresis (CE) and micellar electrokinetic chromatography were used for the separation of widely different compounds from natural materials including antibiotics, humic substances, flavonoids, isoflavonoids, illicit drugs, coumarins, alkaloids, steroids, Chinese herbal preparations, nicotine, caffeine, amphetamines, toxins such as aflatoxins B1, B2, G1, G2, mycotoxins, heptapeptide toxins and others, ephedrine compounds, mineral elements, and natural compounds in biological samples. A discussion of sample extraction and clean-up and the advantages of using CE is also presented.

Animals↗
PubMed1997

Frequency of the variant allele CYP2D6(C) among North American Caucasian lung cancer patients and controls.

Previous reports of the association between the debrisoquine polymorphism and lung cancer risk are conflicting. Following the report of an association between lung cancer risk and the variant allele CYP2D6(C), we examined the presence of this allele in 98 incident Caucasian lung cancer patients and 110 age, race, and sex matched hospital controls from a case-control study conducted at the National Naval Medical Center in Bethesda, MD. Debrisoquine metabolic phenotype was determined by debrisoquine administration and analysis of debrisoquine and 4-hydroxydebrisoquine in the subsequent 8 h urine collected. Genomic DNA was genotyped by a specific polymerase chain reaction amplification and subsequent restriction enzyme digestion, and Southern analysis. Twenty subjects were heterozygous for the CYP2D6(C) allele but none were homozygous for this allele. There was no significant difference in frequency of CYP2D6(C) between lung cancer patients and controls (5.61% and 4.09%, respectively), and there was no significant heterogeneity among cases by histologic type of lung cancer (P = 0.08). However, 7 of 11 cases (64%) with the CYP2D6(C) allele had small cell lung cancer, and none had squamous cell carcinoma. Carrying the CYP2D6(C) allele did not impair debrisoquine metabolism to the same degree as the known inactivating mutations, CYP2D6(A) and CYP2D6(B), or deletion of CYP2D6. Thus, the CYP2D6(C) allele does not encode a completely inactivating mutation, and the suggestion of a role for this variant allele in the risk for specific histologic types of lung cancer justifies further investigation.

Alleles↗
PubMed1996

High-speed screening of polymerase chain reaction products by capillary electrophoresis.

In an effort to develop capillary electrophoresis (CE) for high-throughput polymerase chain reaction (PCR) molecular diagnostics, a method was developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR products (375 and 400 bp) in an assay of TGF-beta 1 knockout mice to determine the genotype of neonates. Using a commercially available CE instrument, the two PCR products were separated in 12 min with a replaceable gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm field strength. With the coinjection of a 20-bp ladder, the sizes of the PCR products were determined from the electropherogram without using a calibration plot and curve-fitting program. Faster separation was obtained using the combination of a short effective length capillary and high field strength. The two PCR products were separated in 82 s with a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separation, with minimum postrun data processing, is highly desirable for the high-throughput screening of PCR products using a single-capillary CE system.

Animals↗
PubMed1996

Micellar electrokinetic chromatography in zero-electroosmotic flow environment.

Micellar electrokinetic chromatography (MEKC) is conducted in polyacrylamide-coated capillaries under almost complete suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of MEKC operation are presented. The technique is termed reversed-flow MEKC (RF-MEKC) because, in contrast to MEKC in bare-silica capillaries (N-MEKC), solute migration order is reversed and solute migration time is inversely proportional to micelle concentration. This presents an advantage for the high-efficiency separation of extremely and moderately hydrophobic solutes in a short analysis time. Examples of the separation of polycyclic aromatic hydrocarbons, aflatoxins and dansylated-amino acids are presented using sodium dodecyl sulfate (SDS) surfactant. Polycyclic aromatic hydrocarbons are separated using a relatively low micelle concentration. The detection sensitivity for these compounds is enhanced in two ways. First, the peaks are sharp because of the short analysis time and the inertness of the column surface. Second, the fluorescence background and Joule's heating are minimal because of the low concentration of SDS and other additives needed to affect the separation. While N-MEKC is mainly conducted with basic buffers, RF-MEKC can be conducted in basic as well as acidic media as illustrated in the separation of 15 dansylated-amino acids at pH 4.2.

Chromatography, Liquid↗
PubMed1996

Electrokinetic chromatography in suppressed electroosmotic flow environment: use of a charged cyclodextrin for the separation of enantiomers and geometric isomers.

Electrokinetic chromatography (EKC), with negatively-charged cyclodextrins (NCDs) added to the buffer, was conducted in polyacrylamide-coated columns under suppression of electroosmotic flow. The equations of migration and resolution for neutral solutes in this mode of chromatography, which for brevity we term NCD-EKC, are presented. The chiral sulfated cyclodextrin, beta-CD-SBE (IV), used in this study is anionic over the entire pH range accessible to capillary electrophoresis, and the coated columns are stable and provide reproducible performance in the pH range 2.5-8.8. Optimum separation was obtained in the pH range where the solutes are neutral. The incorporation of an alkyl spacer between the sulfate ion and the rim of the cyclodextrin allows an unhindered approach and inclusion of neutral solutes in the cyclodextrin cavity. Solute migration time is inversely proportional to the concentration of the chiral selector. Separation (relative migration time difference) increases with decreasing chiral selector concentration and approaches a maximum, beyond which further decreases in chiral selector concentration result in broad peaks and loss of resolution. A chiral selector concentration of 1% in a 10 mM phosphate buffer produced excellent separation of amino acids and dipeptide enantiomers. In addition to being chiral selectors, cyclodextrins are also known as shape selectors. NCD-EKC is particularly suited for the separation of positional isomers of hydrophobic solutes. The separation of aflatoxin isomers and chlorophenol congeners is presented. In the separation of chlorophenols the more hydrophobic trichlorophenols eluted first and the least hydrophobic, phenol, eluted last.

Amino Acids↗
PubMed1995

Separation of tryptophan and related indoles by micellar electrokinetic chromatography with KrF laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) was applied for the separation of tryptophan and related indoles. Using a 5 mM sodium borate buffer (pH 9.2) containing 50 mM sodium dodecyl sulfate and 5% acetonitrile, eleven indoles were baseline separated in under 17 min. Most of the indoles were detected at the nM level by native fluorescence using KrF laser-induced fluorescence (LIF), which was approximately 100 times more sensitive than UV absorption detection at 200 nm. Preliminary results show that the MEKC-LIF with direct sample injection is a feasible method for assessing indole profiles in diluted urine and serum.

Electrophoresis, Capillary↗
PubMed1995

Separation and detection of acidic and neutral impurities in illicit heroin via capillary electrophoresis.

The separation and detection of acidic and neutral impurities in illicit heroin using capillary electrophoresis (CE) is described. Separations were achieved using charged cyclodextrin modified micellar electrokinetic capillary chromatography. The use of the anionic beta-cyclodextrin sulfobutyl ether 1V in combination with sodium dodecyl sulfate significantly increased resolution. Improved selectivity and/or sensitivity in detection was obtained using photodiode array ultraviolet and laser-induced fluorescence detection. The phenanthrene-like heroin impurities exhibit high native fluorescence under krypton-fluoride laser excitation (248 nm). The limit of detection by laser-induced fluorescence detection for one of these solutes (acetylthebaol) is 1.8 ng/ml, 500 times more sensitive than UV. This methodology is applicable to analysis of both crude and refined heroin.

Drug Contamination↗
PubMed1995

Separation of estrogens by micellar electrokinetic chromatography.

Capillary electrophoresis of the sex hormone estrogens using different buffer components was investigated. Free zone electrophoresis with 10 mM phosphate buffer (pH 11.5) or 10 mM phosphate buffer with 10-20% methanol was not effective in separating the ten estrogens used in this study. However, nine estrogens were resolved by micellar electrokinetic chromatography using a 10 mM borate buffer (pH 9.2) containing 100 mM sodium cholate. In addition, some estrogens were partially separated using sodium dodecyl sulfate (SDS) micellar buffers; however, the addition of modifiers such as organic solvents or cyclodextrins improved resolutions significantly. Using a 10 mM phosphate buffer (pH 7.0) containing 50 mM SDS and 20% methanol, or a 10 mM borate buffer (pH 9.2) containing 50 mM SDS and 20 mM gamma-cyclodextrin, all ten of the tested estrogens were separated. However, the cyclodextrin-modified buffer allowed faster separation.

Chromatography, Liquid↗
PubMed1995

Separation and detection of amino acids and their enantiomers by capillary electrophoresis: a review.

Since its introduction as an analytical technique capillary electrophoresis has been used for the separation of amino acids and their enantiomers; over 150 studies have been published to date. This review deals with their separation and detection. Amino acids have been resolved using both capillary zone electrophoresis and micellar electrokinetic chromatography. Pre-column derivatization schemes which are employed for the sensitive detection of amino acids are discussed. Criteria for the selection of the pre- or post-column derivatizing agent, chromophore or fluorophore, are presented. Detection systems, direct and indirect, that have been used are given with emphasis on fluorogenic reagents and laser induced fluorescence detection. Also, procedures for the separation of amino acid enantiomers are discussed and illustrated.

Amino Acids↗
PubMed1995

Enantiomeric separation of amino acids using micellar electrokinetic chromatography after pre-column derivatization with the chiral reagent 1-(9-fluorenyl)-ethyl chloroformate.

Direct enantiomeric separations of some racemic amino acids derivatized with 9-fluorenylmethyl chloroformate were obtained using cyclodextrin-modified micellar electrokinetic chromatography (CD/MEKC) with a buffer made up of 5 mM sodium borate (pH 9.2), 150 mM sodium dodecyl sulfate (SDS) and 40 mM gamma-CD. Alternatively, enantiomeric separations were also achieved indirectly using MEKC after pre-column derivatization with (+)-1-(9-fluorenyl) ethyl chloroformate (FLEC). Using either a 10 mM sodium phosphate (pH 6.8) or a 5 mM sodium borate buffer (pH 9.2), each of which contained 25 mM SDS and 10-15% of acetonitrile, FLEC-derivatized serine, alanine, valine, methionine, leucine, phenylalanine, tryptophan, and their diastereomeric pairs were all separated: the L-isomers migrated faster than the corresponding D-isomers. However, when (-)-FLEC was used for derivatization, the D-isomers migrated faster than the corresponding L-isomers. Also, the diastereomers of aspartic acid, glutamic acid, and proline were resolved using a 10 mM sodium citrate buffer (pH 4.4). Using KrF (248 nm) laser-induced fluorescence, the detection limit of (+)-FLEC derivatized DL-amino acids was obtained at the nM level, which was about 100 x more sensitive than UV absorption at 200 nm. Analyte concentrations as low as 3 x 10(-8) M (DL-Val) could be derivatized with (+)-FLEC.

Acetonitriles↗
PubMed1994

Analysis of nitrate and nitrite in water and urine by capillary zone electrophoresis.

A capillary zone electrophoresis method for the separation and analysis of nitrate and nitrite in water and urine was developed. No interference in the electropherogram from other anions is observed by using a polyacrylamide-coated column with a modified phosphate buffer at pH 3 for the separation, and UV absorption at 214 nm for the detection. The method does not require sample pretreatment or the use of organic solvents. The limit of detection for each analyte (S/N = 3), using a 75 microns I.D. capillary, is 0.5 microgram/ml. Urine samples require 40-fold dilution in order to maintain migration time reproducibility to within 1% relative standard deviation.

Electrophoresis↗
PubMed1994

High-performance liquid chromatography and micellar electrokinetic chromatography of taxol and related taxanes from bark and needle extracts of Taxus species.

High-performance liquid chromatography (HPLC) and micellar electrokinetic chromatography (MEKC) were applied for the separation of taxol, cephalomannine, and baccatin III in crude extracts from the needle and bark of Taxus species. The chromatogram of the bark extract was cleaner than that of the needle allowing a more reliable detection of taxol and cephalomannine in the bark extract. However, HPLC quantitation of taxol in the needle extract would be difficult due to coeluting taxinines. Nevertheless, this was not a problem in the MEKC experiment. In comparison to HPLC, MEKC offered baseline resolution of taxol from taxinines in the needle extract, less solvent waste, a smaller sample requirement, and the simultaneous detection of taxol, cephalomannine and baccatin III in a relatively simpler electrophoretic run.

Alkaloids↗
PubMed1994

Micellar electrokinetic chromatography of the anti-human immunodeficiency virus (HIV) agent michellamine B with absorption and laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) was applied to the separation of the anti-HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium deoxycholate and 10-20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high-performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser-induced fluorescence detection (LIF). Using the sodium dodecyl sulfate-containing buffer and LIF, the limit of detection for michellamine B was approximately 2 ng/mL. The sensitivity was degraded approximately 100-fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.

Antiviral Agents↗
PubMed1994

Accumulation and persistence of individual polychlorinated biphenyl congeners in liver, blood, and adipose tissue of rats following dietary exposure to Aroclor 1254.

Female F344/NCr rats were exposed continuously to Aroclor 1254 (1, 3.3, 10, 33, or 100 ppm in the diet) for 7, 28, or 84 days in order to assess the accumulation of polychlorinated biphenyls (PCBs) in liver, blood, and adipose tissue. The persistence of the individual PCB congeners which are detected in liver was examined in the three tissues of additional groups of rats exposed for 7 days followed by 21 days on control diet, or for 28 days followed by 56 days on control diet. Limited accumulation of PCB congeners with low chlorine substitution (tri- and tetrachlorobiphenyls) in the liver and blood, and preferential retention of highly substituted PCB congeners (penta- and hexachlorobiphenyls) were observed in rats continuously exposed to Aroclor. In these rats, time- and dose-dependent increases in the relative levels of two congeners which cause profound phenobarbital-type induction [2,2',3,4,4',5'-hexachlorobiphenyl (BZ# 138) and 2,2',4,4',5,5'-hexachlorobiphenyl (BZ# 153)] were detected in the liver and adipose tissue. Rats receiving control diet following Aroclor treatment displayed a time- and dose-dependent decrease in the relative levels in blood, adipose and hepatic tissue of 2,3,3',4,4'-pentachlorobiphenyl (BZ# 105) and 2,3',4,4',5-pentachlorobiphenyl (BZ# 118), two of the major congeners showing both TCDD- and phenobarbital-type induction. These rats also displayed increases in the relative adipose levels of another relatively potent mixed-type inducer, 2,3,3',4,4',5-hexachlorobiphenyl (BZ# 156), and increases in adipose and hepatic levels of the pure phenobarbital-type inducer, 2,2',4,4',5-pentachlorobiphenyl (BZ# 99).

Adipose Tissue↗
PubMed1994

Promotion by polychlorinated biphenyls of lung and liver tumors in mice.

Polychlorinated biphenyls (PCB), which are tumor promoters, have been found in human tissues for decades. Their contribution to cancer risk may only now start to appear, due to long human cancer latency and the nature of tumor promotion. Epidemiological associations have been seen between PCB exposure or tissue content and cancer at several sites. In rodents, tumor promotion by PCBs has been little studied in tissues other than liver. Previously, in an experiment modeling infant carcinogen exposure following PCBs received in milk, lung and liver tumors, initiated neonatally in mice by the environmental nitrosamine N-nitrosodimethylamine (NDMA), were promoted by later treatment with Aroclor 1254. The present study was undertaken to confirm and characterize the effects of Aroclor 1254 on tumor number, latency, size and malignancy. Male Swiss mice were given NDMA on postnatal day 4 and Aroclor 1254 (250 mg/kg) on day 8, and killed at intervals. Eight PCB congeners were quantified in the carcasses. Incidences of mice with NDMA-initiated lung tumors at 28 weeks of age were increased 2.5-fold by PCBs. Multiplicities of lung tumors were enhanced four-fold by PCBs at 28 and 52 weeks. By 72 weeks tumor numbers were similar in the NDMA-only and NDMA-PCB groups. Liver tumors first occurred in significant numbers at 52 weeks and only in mice receiving both NDMA and PCBs. As for the lung, at 72 weeks the incidence was high in both the NDMA-only and NDMA-PCB groups. Sizes of tumors and liver carcinoma incidence were not altered by PCB treatment. Carcass analysis revealed a significant positive association between lung tumor numbers at 28 weeks and relative percentage of 2,2',4,4',5-pentachlorobiphenyl, with no other correlations. The results confirm that PCBs promote lung as well as liver tumors, by triggering the early appearance of latent initiated tumors otherwise presenting in old age.

Animals↗