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Manipulating biological samples for environmental scanning electron microscopy observation.

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.

Animals↗

PubMed2000

Use of nitric acid in sample pretreatment for determination of trace elements in various biological samples by ETAAS.

Trace elements in liquid biological samples may be determined by direct electrothermal atomic absorption spectrometry (ETAAS). In our previous work it was found that samples containing proteins or DNA may leak out of the graphite tube before the drying step, despite the addition of various modifiers. In order to keep the sample to the graphite tube, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid, or 5 microl of 32% v/v nitric acid was added to the graphite tube before ETAAS determination. Applying the proposed procedure, the concentrations of lead in eluted fractions after gel chromatographic separation of human cerebellar nucleus dentatus supernatant and platinum in isolated DNA samples were determined. The use of nitric acid in sample pretreatment prevent sample leakage out of the graphite tube, provided for even drying and considerably reduced nonspecific absorption in lead determination. The repeatability of measurements was better than + 6%. The accuracy of the procedure was checked by spiking samples. The recoveries for both elements lay between 93--104%. Nitric acid was found to be a better modifier than TRITON X-100.

Calibration↗

PubMed2003

[Study on the level of phthalates in human biological samples].

OBJECTIVE: To monitor the level of phthalates in human biological samples. METHODS: The concentrations of three commonly-used phthalate (di-ethyl phthalate, DEP; di-n-butyl phthalate, DBP; di-2-ethylhexyl phthalate, DEHP) in the human biological samples were measured by using reversed-phase HPLC. The blood serum samples were collected from 52 women and 8 men, semen specimens from 36 men, and fat samples from 6 women and 5 men. All these people were randomly selected, from 23 to 50 years of age and residing in Shanghai. We also measured hormone levels of serum and conventional indices of semen specimens. RESULTS: The three phthalates were detected in most of the biological samples, with median levels of 5.71 mg/L (ND-37.91 mg/L) in blood serum, 0.30 mg/L (0.08 -1.32 mg/L) in semen specimens, and 0.72 mg/kg (ND-2.19 mg/kg) in fat samples. The spearman correlation coefficients between concentrations of phthalates and levels of hormone in serum were 0.442 for DBP and E(2), and -0.486 for DEP and testosterone. There was a positive association between liquefied time of semen and semen concentrations of phthalates. The correlation coefficients were 0.456 for DEP, 0.475 for DBP, and 0.457 for DEHP, respectively. There was no significant difference between semen concentrations of phthalates and sperm density. CONCLUSION: These results suggest that people residing in Shanghai area are exposed to phthalates (particularly to DBP and DEHP) though the level is still relatively low.

Adipose Tissue↗

PubMed1996

Microwave digestion using dual PTFE containers for analysis of trace elements in small amounts of biological samples.

The analysis of trace elements in biological samples is essential to extend our knowledge on human health and disease. Inductively coupled plasma mass spectrometry (ICP-MS) makes it possible to simultaneously determine these elements in trace amounts. Before analysis, however, biological samples such as organs and tissues must be liquefied and extra organic materials must be decomposed by acid digestion. We established a method of microwave digestion using dual PTFE containers to minimize the amount of samples. Samples (35-45 mg) of standard reference materials, bovine liver (1577a, NIST) and fish flesh (MA-A-2, IAEA), were weighed in PTFE-PFA vials and a small amount of nitric acid (0.5 ml) was added. The vials were sealed and two PTFE-PFA vials were placed in a PTFE-TFM vessel containing 6 ml of pure water. Then the vessels were placed in a rotor and the samples were digested for 38 min in a microwave oven according to a pre-set program. After the program was completed, the samples were analyzed by ICP-MS. The determined values of elements of the microwave-digested samples matched the certified values of the standard reference materials. Therefore, the digestion using dual containers was successfully applied to small samples.

Animals↗

PubMed2006

Research on stored biological samples: views of African American and White American cancer patients.

Proposals on consent for research with biological samples should be informed by empirical studies of individuals' views. Studies to date queried mostly white research subjects. The aim of this study was to compare the views of two groups of patients: cancer patients at a university clinic (Winship Cancer Institute at Emory Healthcare) and cancer patients at an inner city county hospital (Grady) who were given the option of tissue banking. Overall, 315/452 (70%) patients completed the survey. The Grady cohort was 86% African American; the Winship cohort was 82% White. The vast majority (95%) of individuals in both cohorts agreed to provide a biological sample for future research. Both cohorts were willing for their samples to be used to study cancer and other diseases, including Alzheimer disease. Few participants preferred to control the disease to be studied (10%) or wished to be contacted again for consent for each future research project (11%). In our sample, almost all clinical patients, regardless of site of care, ethnicity or socioeconomic status, were willing to provide a biological sample for research purposes and allow investigators to determine the research to be done without contacting the patients again. These findings support the recommendation to offer individuals a simplified consent with a one-time binary choice whether to provide biological samples for future research.

Black or African American↗

PubMed2006

Capillary electrophoresis screening of poisonous anions extracted from biological samples.

A method was developed for screening human biological samples for poisonous anions using capillary electrophoresis (CE) employing indirect UV detection. The run buffer consisted of 2.25 mM pyromellitic acid, 1.6 mM triethanolamine, 0.75 mM hexamethonium hydroxide and 6.5mM NaOH at pH 7.7. Biological samples were pretreated using solid phase extraction. The method was applied to the analysis of human blood, plasma, urine, and intestinal contents. Twenty-nine different anions were detectable at aqueous concentrations of 1 part per million (ppm) with a typical analysis time less than 20 min. Intraday migration time R.S.D. and peak area R.S.D. for blood samples were less than 1.1% and 6.3%, respectively. Interday migration time R.S.D. for plasma samples ranged from 7.5% to 10.4%. The new method produced efficient separations of various target anions extracted from complex biological matrices.

Acids↗

PubMed2004

Determination of amines as pentafluoropropionic acid anhydride derivatives in biological samples using liquid chromatography and tandem mass spectrometry.

Determination of amines in biological samples as markers of exposure to the amines or the corresponding isocyanates is an important tool for industrial exposure assessment. In this study, a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for determination of amines in biological samples as perfluorofatty amides derivatives is presented. The method enables determination of diamines such as methylene diamine (MDA), toluene diamine (TDA), naphthalene diamine (NDA), hexamethylene diamine (HDA), isophorone diamine (IPDA), methylenedi(cyclohexylamine)(HMDA) and 4,4'-methylene-(2-chloroaniline)(MOCA) in human urine and plasma. The work-up procedure included hydrolysis of the biological samples with 3 M H(2)SO(4) at 100 degrees C for 16 h and extraction of the amines into toluene, where derivatisation of the amines with perfluorofatty acid anhydride was performed. Following removal of excess reagent and the acid formed and an exchange of solvent, the derivatives were analysed using gradient elution with an acetonitrile/water mobile phase composition and electrospray ionisation (ESI) with multiple reaction monitoring (MRM) of [M - H](-)-->[M - H - 120](-) or [119](-). Several perfluorofatty acid anhydrides were evaluated as derivatisation reagents, but the LC chromatographic properties of the pentafluoropropionic acid anhydride (PFPA) derivatives were favourable. Quantification of amine-PFPA derivatives was performed using deuterium labelled amine-PFPA derivatives as internals standards with good precision and linearity in the investigated range of 0-20 ng ml(-1) urine. The instrumental detection limits for the amine-PFPA derivatives were 0.2-3 fmol for MRM of [M - H](-)-->[119](-) and 0.3-8 fmol for [M - H](-)-->[M - H - 120](-). In 10 urine and 6 plasma samples from workers exposed to isocyanates, determination of TDA and MDA as PFPA derivatives was performed using LC-MS/MS and a reference GC-MS method. No significant difference between the two methods was observed.

Amines↗

PubMed1986

Specific absorption rate in electrically coupled biological samples between metal plates.

The specific absorption rate (SAR) in a biological sample irradiated by electromagnetic fields between the metal plates of a transmission line can be altered significantly by the spacing of the metal plates and the distance between neighboring samples. The SAR in spherical biological samples is calculated for a number of neighboring sample arrangements and metal-plate spacings by using the method of images and induced dipole coupling. For a decrease in metal-plate spacing, the derived equations predict an increase in SAR within a sample and a decrease in SAR with a decrease in neighboring-sample spacing. The calculations are compared with measurements made with the aid of an array of 1-in radius metal hemispheres on the lower plate of two parallel plates (thus forming an image system). The hemisphere on which measurements are taken is insulated from the metal plate and is connected via a coaxial center conductor to an HP 3582A spectrum analyzer that measures the voltage and hence the electric field intensity at the hemisphere. Measurements made at a frequency where wavelength is large compared with sample size (48 Hz) are in good agreement with calculations.

Absorption↗

PubMed2005

Research with stored biological samples: what do research participants want?

BACKGROUND: There is widespread disagreement about the type of consent needed for research with stored biological samples. Many believe consent for each future use is required to respect individuals. Others worry this approach may block important research. METHODS: We analyzed 1670 consent forms signed by research participants at the Warren G. Magnuson Clinical Center, National Institutes of Health, between January 1, 2000, and May 31, 2002, that offer options for future research with participants' biological samples. The research participants were healthy volunteers, family members of affected individuals, and individuals with a broad range of medical conditions enrolled in clinical research studies with and without the prospect of direct medical benefit. RESULTS: Overall, 87.1% of research participants given the option chose to authorize future research on any medical condition. More than 85% permitted unlimited future research with their stored biological samples regardless of sex, age, geographic location, or whether the individual was affected by the disease being studied or a healthy volunteer. Only 6.7% of those given the option to refuse all future research did so. Although African Americans were less likely to permit future research, 75.0% of African Americans still authorized unlimited future research with their samples. CONCLUSIONS: Most research participants authorize the unlimited future research use of their biological samples when given the opportunity to do so. These findings suggest that providing research participants with a simple binary choice to authorize or refuse all future research might allow individuals to control use of their samples, simplify consent forms, and allow important research to proceed.

Biological Specimen Banks↗

PubMed2000

Optimal sample preparation conditions for the determination of uranium in biological samples by kinetic phosphorescence analysis (KPA).

Kinetic phosphorescence analysis (KPA) is a proven technique for rapid, precise, and accurate determination of uranium in aqueous solutions. Uranium analysis of biological samples require dry-ashing in a muffle furnace between 400 and 600 degrees C followed by wet-ashing with concentrated nitric acid and hydrogen peroxide to digest the organic component in the sample that interferes with uranium determination by KPA. The optimal dry-ashing temperature was determined to be 450 degrees C. At dry-ashing temperatures greater than 450 degrees C, uranium loss was attributed to vaporization. High temperatures also caused increased background values that were attributed to uranium leaching from the glass vials. Dry-ashing temperatures less than 450 degrees C result in the samples needing additional wet-ashing steps. The recovery of uranium in urine samples was 99.2+/-4.02% between spiked concentrations of 1.98-1980 ng (0.198-198 microg l(-1)) uranium, whereas the recovery in whole blood was 89.9+/-7.33% between the same spiked concentrations. The limit of quantification in which uranium in urine and blood could be accurately measured above the background was determined to be 0.05 and 0.6 microg l(-1), respectively.

Humans↗

PubMed1988

Use of platinum as a modifier in the sensitive detection of tellurium in biological samples.

Estimation of tellurium in biological samples by flameless atomic absorption spectrophotometry is hindered by the high volatility of the metal. This necessitates the use of low ashing temperatures which are inadequate to thoroughly ash the samples and thereby reduce interference due to smoke during the atomization stage. The use of platinum as a chemical modifier to thermally stabilize tellurium has, therefore, been explored. Thermal stability of tellurium was dependent on the concentration of platinum; maximum enhancement in stability was achieved at a platinum concentration of 10 microgram/ml or greater, which allowed ashing temperatures to be increased from 400 to 1300 degrees C. A threefold increase in the sensitivity for tellurium determination was also obtained in the presence of platinum. The thermal stability and the sensitivity, however, were susceptible to the presence of organic, inorganic, and biological matrices. This procedure for the determination of tellurium, stabilized probably in the form of an amalgam with platinum, has been used successfully to estimate tissue levels of the metal following administration to mice of a novel tellurium-containing immunostimulant agent. Detection limits in urine, plasma, and tissues were about 50, 5, and 170 ng of tellurium per milliliter or gram, respectively.

Animals↗

PubMed1991

Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples.

D-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of D-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, D,L-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for D-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for D-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine D-lactate in blood plasma volumes of less than 1 microliter.

Animals↗

PubMed2006

Stability of glufosfamide in phosphate buffers and in biological samples.

Glufosfamide is a new, potential chemotherapeutic agent currently under investigation. Stability of glufosfamide was investigated in sodium phosphate buffers with different pH and temperature and in biological samples. Glufosfamide and isophosphamide mustard were quantified simultaneously using a liquid chromatography-ion trap mass spectrometric method; precision and accuracy were within 15% for each analyte. Glufosfamide was stable in neutral buffers, but decomposed to form isophosphoramide mustard under acidic and basic conditions, which was pH- and temperature-dependent. The stability of glufosfamide varied in different biological samples. Results indicated that glufosfamide was unstable in some biological samples, such as the small intestine, smooth muscles, pancreas and urine, especially in the small intestine homogenate, with a half-life of 1.1 h. But the pH (<8) and beta-glucosidase of the tissue homogenate was found to have negligible contribution to the degradation of glufosfamide. The enzymatic inhibition experiment with the specific inhibitor, saccharo-1,4-lactone, demonstrated that it was glucuronidase that resulted in the degradation of glufosfamide in small intestine homogenate. Methanol was recommended to be used to homogenize the tissue in an ice water bath, and the container for urine collection should also be maintained in an ice water bath, and all the biological samples collected should be preserved in frozen condition until analysis.

Animals↗

PubMed1999

A one-step method for protein estimation in biological samples: nitration of tyrosine in nitric acid.

A number of methods are commonly employed for the determination of protein in biological samples. Unfortunately, several compounds that are constituents of biological buffers interfere with these methods, limiting their application. Previous studies have demonstrated that tyrosine rapidly undergoes nitration in nitric acid to yield 3-nitrotyrosine, which has a lambdamax of 358 nm. Utilizing this reaction, we have developed a one-step method for the assessment of protein content in biological samples. Common interfering substances, including SDS, urea, glycerol, ammonium sulfate, and beta-mercaptoethanol, do not interfere with this method. Because of its simplicity, this reaction might be useful for estimating protein content in a variety of biological samples.

Evaluation Studies as Topic↗

PubMed2002

Determination of ethanol in biological samples by gas chromatography with an electron-capture detection.

A simple and sensitive procedure for determination of ethanol in biological samples was established. In this procedure, ethanol in biological samples was first converted to acetaldehyde by yeast alcohol dehydrogenase. Then, acetaldehyde formed was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with an electron-capture detection. Ethanol concentration in 1 ml of rat blood plasma can be measured as 19.5 nmol/ml with 98.0% recovery. Since this procedure enable to determine minute amount of ethanol in biological samples, the method is useful to study the metabolism of ethanol.

Animals↗

PubMed2002

The debate over research on stored biological samples: what do sources think?

BACKGROUND: The debate over informed consent for research on stored biological samples has enormous scientific implications. Unfortunately, there are no data on individuals' attitudes regarding when their consent should be obtained for such research. METHODS: Data were gathered using a telephone survey of 504 individuals living in the United States. Two cohorts were studied: (1) individuals who had participated in clinical research and contributed biological samples and (2) randomly selected Medicare recipients. RESULTS: Of the respondents, 65.8% would require their consent for research on clinically derived, personally identified samples; 27.3% would require it for research on clinically derived samples that are "anonymized." For research-derived samples, 29.0% of the respondents would require their consent if the samples retain personal identifiers; 12.1% would require it if the samples are anonymized before the research is conducted. Also, 88.8% would want to be informed of results of uncertain clinical significance, and 91.9% would not impose greater safeguards on future research on a different disease. CONCLUSIONS: Current practice and policy recommendations regarding research using stored biological samples may be inconsistent with sources' preferences in several respects. In particular, it appears that most sources want to control whether their samples are used for research purposes, are not concerned with the particular disease that will be studied, and want to receive results of uncertain clinical significance. Follow-up research will be needed to assess the generalizability of the current data.

Aged↗

PubMed1994

Immunoassay for native enzyme quantification in biological samples.

In order to detect low levels of enzyme activity, specifically glucose oxidase, in biological samples, an immunoenzymatic assay was developed since currently available methods could not be used because of either their lack of sensitivity or the conditions prevailing in our samples: turbidity of the medium, presence of redox systems other than glucose oxidase, and high concentration of proteins. The principle of the method is to coat a polystyrene surface with a fragment Fc-specific anti-IgG, then with an antibody directed against the looked-for enzyme, which is simultaneously the antigen and the enzyme activity required for immunoenzymatic detection. We applied this concept to biological samples after glucose oxidase administration to mice. This method achieves specificity and sensitivity (20 ng/mL or 1 ng) with samples of biological origin. No marker is needed since the antigen itself possesses an enzyme activity. This method, which requires a small sample volume (50 microL, 20 microL, if necessary), can be extended easily to the many enzymes currently used as markers. It could also be applied to the native enzymes of medical interest for which antibodies and a colorimetric reaction are available.

Animals↗

PubMed1988

Quantitative microanalysis of bile acids in biological samples. Collaborative study.

The analysis of bile acids in biological samples has always presented a problem because of their complex nature and low concentration. Recently, newer analytical procedures for bile acids have become available, including enzymatic analysis, radioimmunoassay, thin-layer chromatography (TLC), gas chromatography, high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). However, they differ greatly with respect to specificity, sensitivity, accuracy and simplicity. On the other hand, the choice of analytical procedure differs according to the specific aims and the nature of biological samples to be analysed. These newer procedures have been compared in a double-blind fashion by distributing bile, plasma and urine samples to seven participating laboratories. GC-MS-SIM was found to be the most sensitive and reliable, but it requires other procedures for preliminary clean-up and fractionation steps. Enzymatic analysis is simple and gives small analytical errors but tends to over-estimate plasma bile acids. Radioimmunoassay gives variable results but is useful as a screening procedure for large numbers of plasma samples. TLC gives reliable results for biliary bile acids in experienced hands, except for differentiation between conjugated dihydroxycholanoic acids. HPLC, whether using derivatization or with fixed 3 alpha-hydroxy steroid dehydrogenase detection, is suitable for the analysis of major bile acids in normal human serum but not for the identification of unknown minor peaks.

3-Hydroxysteroid Dehydrogenases↗