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PubMed1979

The dynamics of IgG in the cornea.

Fluoresceinated IgG was injected centrally into the rabbit's cornea and both its rate of loss from the tissue and its rate of spread in the stroma were estimated by objective fluorometry. A new technique for determining the rate of loss was established which required only the measurement of the maximum fluorescence in the tissue. The rate at which freshly formed IgG in the blood would arrive at the central regions of the cornea was estimated for the human eye. As penetration continues from the limbus and the protein is lost to the aqueous humor, a standing concentration gradient is set up with a minimum at the corneal center. Calculation suggests that this gradient should be small for IgG in the human eye.

Animals↗
PubMed1976

Chromosomal studies of human prostatic tumors in vitro.

Chromosomal studies were performed on primary cultures from human prostatic adenomas and carcinomas. All cells from both tumor types showed diploid chromosomal counts with a normal human karyotype. Chromosomal analysis was also performed on cells of the permanent epithelial line EB 33 derived from a human prostatic carcinoma in this laboratory. In contrast to the primary cultures, hypotriploid chromosome numbers were found during continuous culture for more than 1 year. The range of chromosome numbers increased with time in culture and was reduced to the original model number of 64 by only one animal passage ("nude" mice). Feulgen fluorometry on different passages of the permanent cell line EB 33 showed tetraploidy. This discrepancy to the hypotriploid chromosome number can be explained by different Feulgen hydrolytic properties of the different cell types.

Animals↗
PubMed1983

Liquid-chromatographic determination of indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human plasma.

We describe a "high-performance" reversed-phase liquid-chromatographic method for determination of indole-3-acetic acid (I) and 5-hydroxyindole-3-acetic acid (II) in human plasma. I is eluted at 1.0 mL/min with a mixture of 1-pentanesulfonic acid (pH 3.1), methanol, and water. It is detected by fluorometry. A mixture of citric acid/sodium phosphate solution (pH 4.8) and methanol, at 1.5 mL/min, is used to elute II, which is detected with an electrochemical cell. Platelet-poor plasma samples were pretreated with HCl, perchloric acid, and trichloroacetic acid for protein precipitation. Best results were obtained with the last (protein precipitation is incomplete with HCl, while recoveries of I are concentration dependent with perchloric acid). Analytical recoveries were 58% (SD 3.1%, CV 5.3%, n = 12) and 79% (SD 3.3%, CV 5.3%, n = 9) for I and II, respectively. Concentrations of I and II in plasma ranged from 0.61 to 3.32 (mean 1.54, SD 0.59, n = 15) mumol/L and from 33.0 to 102.6 (mean 51.8, SD 20.1, n = 16) nmol/L, respectively.

Adult↗
PubMed1984

Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence.

Using multifrequency phase and modulation fluorometry and a nonlinear least-squares analysis of lifetime data, we were able to determine the complex decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in synthetic phospholipid bilayers. Our results showed a monoexponential decay of DPH in the pure isotropic solvents studied, over a wide temperature range, and a double-exponential decay of DPH in phospholipids, both above and below the transition. During the transition, and in mixed-phase phospholipids, a three-component analysis was successfully accomplished, and the pre-exponential factors of the two main components have been shown to be quantitatively representative of the gel and liquid-crystalline phases of the bilayer. The fractional intensity of the shorter lifetime component depends on the modalities of the sample preparation. The factors affecting this component are discussed. From the DPH fluorescence lifetime and from the anisotropy data in L-alpha-dimyristoyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidyl choline mixtures, a phase diagram was independently constructed. Conclusions about the sensitivity and the partition of the probe between gel and the liquid-crystalline phases of the bilayer are derived. Lifetime experiments on DPH in a L-alpha-dilauroyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidylch oline mixture suggested a general method for the determination and quantitation of the two different phases in the bilayer.

Dimyristoylphosphatidylcholine↗
PubMed1983

New techniques and tools for clinical chemistry.

In this paper are described and evaluated several new tools of potential use in clinical chemistry. The first, intended to minimize required sample volumes, is a device with which a total sample volume of 1 microL can be dispensed in the form of 1000 identical aliquots. Any number of such nanoliter aliquots can be taken if larger samples are needed. The second new tool is one for detecting anions or cations separated by ion chromatography. Unlike conventional conductometric detectors used in ion chromatography, the new system offers potential sensitivities in the sub-microgram per liter range and useful operating ranges up to 100 mg/L. The third tool is a scheme for background correction in atomic absorption spectrometry; the new technique requires no special auxiliary sources or double-beam optics. Finally, fluorescence time-decay curves and fluorescence lifetimes are shown to be able to overcome the effects of diffusional quenching and scattering resulting from turbidity of solutions in clinical fluorometry.

Chemistry, Clinical↗
PubMed1980

Estimation of phosphatidylglycerol in fluids containing pulmonary surfactant.

A simple, direct and non-destructive method for quantitative separation of phosphatidylglycerol from other phospholipids in pulmonary washings is described. Total lipid extracts from dog lung washings and phosphatidylglycerol standard were spotted quantitatively on chromatoplates and separated by one-dimensional thin-layer chromatography in chloroform-methanol-water 65:25:4 (v/v/v). Quantification was performed with Rhodamine 6G and fluorometry. Washings from eleven dogs contained (mean +/- S.E.) 236 +/- 25 microgram phosphatidylglycerol per g of parenchymal wet lung tissue which accounted for 8.7% +/- 2.3 of the total phospholipids. The procedure is especially useful for quantification of phosphatidylglycerol in microgram amounts.

Animals↗
PubMed1980

Fluorometric and colorimetric enzymic determination of triglycerides (triacylglycerols) in serum.

We describe two fully enzymic methods, fluorometric and colorimetric, for determination of triglycerides (triacylglycerols) in serum. Samples are incubated with microbial lipase for 10 min, and the glycerol released from the triglycerides is oxidized by NAD+ in the presence of glycerol dehydrogenase. In the fluorometric method, the resulting NADH is in turn oxidized by resazurin as catalyzed by diaphorase to form resorufin, a highly fluorescent compound. In the colorimetric method, the NADH is oxidized by coupling with a tetrazolium salt/diaphorase system to form formazan, a highly colored compound. Calibration curves, constructed by plotting change in fluorescence or absorbance vs concentration of triglycerides, were linear up to 6 and 5 g of triglycerides per liter of serum for the fluorometric and colorimetric methods, respectively. The assays require only 5 and 15 microL of serum for fluorometry and colorimetry, respectively. The CV was 0.59% for the fluorometric method, 0.91% for the colorimetric procedure. The time for analysis for either method is less than 15 min. The results correlate well with those obtained by the Dow Diagnostic Kit method, a colorimetric method in which glycerol kinase and glycerol-1-phosphate dehydrogenase form NADH from ATP and NAD+ in the presence of glycerol and glycerol 1-phosphate.

Colorimetry↗
PubMed1980

Cardiotoxicity and comparative pharmacokinetics of six anthracyclines in the rabbit.

Six anthracycline antibiotics with demonstrated antitumor activity in human or experimental tumor systems were studied. The purpose of this investigation was to compare the cardiotoxic potential of these compounds and to characterize the myocardial pharmacokinetics in order to provide a possible explanation for differences in cardiotoxicity. Groups of rabbits received i.v. injections of drug at maximally tolerated treatment doses with respect to lymphohematopoietic toxicity for periods of 11 or 16 weeks and were evaluated histopathologically for the development of myocardial damage. Following a single i.v. administration of the different anthracyclines to rabbits, the amount of parent drug and metabolites accumulating in the heart at various times was determined by high-pressure liquid chromatography and fluorometry. Adriamycin (ADR), daunorubicin (DNR), and detorubicin produced similar severe cardiomyopathy with frequent congestive heart failure at approximately equal dose levels. Three additional antibiotics, rubidazone and the N-L-leucyl derivatives of ADR and DNR (N-L-leucyl-adriamycin and N-L-leucyl-daunorubicin), produced significantly less severe lymphohematopoietic toxicity, thus permitting the administration of 3 to 3.5 times the ADR and DNR treatment doses. Chronic treatment with these anthracyclines also resulted in significantly less cardiomyopathy, especially in the case of N-L-leucyl-daunorubicin and rubidazone. This reduced cardiomyopathy correlated with lower total myocardial drug accumulation but, more importantly, with lower amounts of DNR or ADR accumulation in the heart. These findings suggest that the degree of anthracycline myocardial toxicity may be directly related to the relative qualitative and quantitative accumulation of drug metabolites in the myocardium.

Animals↗
PubMed1982

Intracellular adriamycin levels and cytotoxicity in adriamycin-sensitive and adriamycin-resistant P388 mouse leukemia cells.

Adriamycin (ADR) (NSC-123127) uptake and retention in ADR-sensitive P388 leukemia (P388/S) and ADR-resistant P388 leukemia (p388/R) cells were compared by fluorometry and laser flow cytometry (FCM) and were correlated with cytotoxic effects. Drug levels in P388/R cells treated in vitro with ADR (1-10 micrograms/ml) were twofold to fourfold lower than were levels in similarly treated P388/S cells FCM analysis of P388/S and P388/R cells exposed in vitro to ADR showed qualitative and quantitative differences in ADR fluorescence profiles of drug-treated cells (1-5 micrograms/ml) but not of the isolated nuclei (0.5- 10 micrograms/ml). Drug-induced perturbations in cell cycle traverse and chromosome aberrations were seen in P388/S but not in P388/R cells treated with 0.5-5 micrograms ADR/ml in vitro or 4-8 mg ADR/kg in vivo. The role of FCM in rapidly comparing and quantitating cellular ADR fluorescence profiles of ADR-sensitive and ADR-resistant tumors was demonstrated.

Animals↗
PubMed1980

Analysis and enrichment of murine natural killer cells with the fluorescence-activated cell sorter.

The antiserum anti-NK 1.1 defines an alloantigen specific for natural killer (NK) cells on normal C57BL/6 spleen cells (SC). With complement this antiserum lysed an insignificant percentage of SC, yet could deplete SC suspensions of NK effector cells. The antiserum was also used in this study to indirectly fluorescein label NK cells. The anti-NK 1.1 serum labeled 10 to 15% of nonadherent, nylon column passed SC. The labeled cells were analyzed on the fluorescence-activated cell sorter (FACS), by using flow fluorometry, and were found to be small to medium-sized cells. SC were sorted on the FACS into labeled (NK-1+) and unlabeled (NK-1-) populations, and assayed for NK activity on YAC-1 cells. When compared with control SC, labeled NK-1+ cells were enriched 4- to 13-fold in lytic activity, whereas unlabeled NK-1- cells had little if any NK effector function. Thirty to 60% of the labeled SC adhered to YAC-1 tumor cells in a visual target binding cell assay. The percentage of lymphocytes in the sorted NK-1+ population that bound YAC-1 cells was over 3-fold greater than in unsorted control SC preparations. NK-1- sorted cells did not bind YAC-1 targets. In a preliminary experiment NK-1+ sorted SC inhibited outgrowth of YAC in A/J mice, whereas NK-1- sorted cells did not.

Animals↗
PubMed1982

A high-pressure liquid chromatographic-enzymatic assay for glycine and taurine conjugates of cholic, chenodeoxycholic and deoxycholic acid in serum.

An assay for glycine and taurine conjugates of cholic, chenodeoxycholic and deoxycholic acid in serum by a high pressure liquid chromatographic-enzymatic system is presented. The bile acids are extracted from serum by a reverse-phase liquid chromatographic process with an octadecylsilane column. The bile acid conjugates are separated on a muBondapak C18 column with methanol/KH2PO4 buffer, 20 mmol/l, pH 5.3 as a mobile phase in less than 30 min at a flow rate of 1.4 ml/min. The bile acid fractions are measured by enzymatic fluorometry using a 3 alpha-hydroxysteroid dehydrogenase-diaphorase system. Recoveries ranged from 82 to 96%, coefficients of variation were from 5 to 15%, and detection limits were from 0.03 to 0.08 mumol/l. Mean serum concentrations ranged from 0.10 to 0.39 mumol/l in the fasting state and from 0.29 to 1.55 mumol/l postprandially in subjects with normal liver function.

Adult↗
PubMed1981

Monitoring tissue elimination of fluorescein with the perfusion fluorometer: a new method to assess capillary blood flow.

Visual assessment of tissue staining after intravenous fluorescein is a common technique for predicting viability of questionably perfused tissue. The development of the perfusion fluorometer has permitted quantification of tissue fluorescein, providing increased precision. This study employed this instrument to calculate fluorescein elimination from rats with and without raised dorsal pedicle flaps. Control animals exhibited homogeneous patterns of fluorescein elimination consistent with first-order kinetics. Elimination in experimental animals was assessed after the animals received full back skin flaps with the cephalad pedicle remaining intact. Three distinct patterns of elimination were noted in each flap. In the cephalad portion, elimination was similar to control. At the caudad end, no elimination was noted. Midflap, fluorescein was eliminated slowly. These elimination patterns predicted ultimate viability 14 days postoperatively, as they correspond to viable, dystrophic, and transitional sections, respectively (P less than 0.001). We conclude that perfusion fluorometry can assess capillary flow in healthy and ischemic tissue by documenting elimination as well as delivery of fluorescein.

Animals↗
PubMed1995

[The assay of enzyme activity in EIA--colorimetric, fluorometric and luminometric method].

The method of enzyme activity related in enzyme immunoassay was described. The detection methods were colorimetric, fluorometric and chemi- or bioluminometric assay for enzyme, and the sensitivity of three detection methods were compared. The order of detection limit was chemi- or bioluminometry > fluorometry > colorimetry. But it is important that the selection of detection method of enzyme will make fit on your desirable sensitivity of analysis.

Alkaline Phosphatase↗
PubMed1995

A desktop, low-cost video fluorometer for quantitation of macromolecules after gel electrophoresis.

After gel electrophoresis, quantification of in-gel fluorescence is sometimes used to assay the macromolecules fractionated. Both procedures and equipment that have the following improvements are presented here for direct video fluorometry of gels used for electrophoresis: comparatively low cost, high ease of use and low consumption of space. This equipment has a linear response to the amount of ethidium-stained DNA that forms a band in an agarose gel.

DNA, Viral↗
PubMed1995

Intracellular calcium transients underlying interval-force relationship in whole rat hearts: effects of calcium antagonists.

OBJECTIVES: Much of the understanding about the cardiac interval-force relationship of the whole heart, including mechanical restitution and postextrasystolic potentiation (PESP), has been inferred from isolated muscle studies. We tested whether results from isolated muscles about intracellular Ca2+([Ca2+]i) transients underlying the interval-force relationship can be substantiated in whole hearts. Additionally, we investigated whether Ca2+ antagonists could alter [Ca2+]i transients underlying mechanical restitution and postextrasystolic potentiation. METHODS: [Ca2+]i transients were studied in isolated perfused rat hearts by surface fluorometry and Indo-1. Using computer-controlled pacing protocols, we performed restitution curves for left ventricular developed pressure and [Ca2+]i (developed pressure and [Ca2+]i plotted as a function of extrasystolic intervals). To quantify restitution curves, we fitted monoexponential functions to plots and analyzed their shift and slope. Then, we used Ca2+ antagonists, low extracellular Ca2+([Ca2+]o) and PESP to modify restitution curves. [Ca2+]i transients in isolated rat hearts were interpreted as Ca2+ released from the sarcoplasmic reticulum. RESULTS: Interval-dependent changes in developed pressure were strongly correlated to interval-dependent changes in the amplitude of [Ca2+]i transients in isolated whole rat hearts. Additionally, nifedipine and low [Ca2+]o led to similar downward shifts but not to a changed slope of restitution curves for [Ca2+]i. On the other hand, PESP increased the slope of restitution curves for [Ca2+]i. Furthermore, the effect of PESP on developed pressure was blunted by high concentrations of Ca2+ antagonists. CONCLUSIONS: The results from isolated muscles about [Ca2+]i transients underlying the interval-force relationship could be substantiated in whole hearts. Additionally, low [Ca2+]i (induced by nifedipine or low [Ca2+]o) decreased the maximal Ca2+ release of the sarcoplasmic reticulum but did not change the release kinetics. On the other hand, PESP presumably accelerated Ca2+ release kinetics of the sarcoplasmic reticulum.

Animals↗
PubMed1995

Triple-label hybridization assay for type-1 diabetes-related HLA alleles.

We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility (*0201, *0302) alleles and two protective (*0301, *0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex (MHC). The test is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybridization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), terbium (Tb) or samarium (Sm) are used. The labeled probes could be detected using time-resolved fluorometry with sensitivities of 1 x 10(7), 3 x 10(8) and 3 x 10(8) molecules, respectively. Cross-reactions were not found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot samples with known DQB1 alleles were analyzed. A 100% agreement with the reference method was reached. Thus, this triple-label hybridization assay proved to be suitable even for detection of a large number of samples.

Alleles↗
PubMed1976

Flow-fluorometric diagnosis of euploid and aneuploid human lymphocytes.

In this paper the potential of flow-fluorometric DNA determination as a convenient and economical alternative to conventional cytogenetics for the diagnosis of aneuploidy in human lymphocytes is explored. By comparing euploid and aneuploid samples, we found that the fluorescence signals emitted from propidium iodide (PI) stained cells are linearly proportional to DNA content. Variation in DNA content between euploid individuals of a given sex was sufficiently low to permit diagnosis of aneuploidy involving chromosomes with greater than 1.8% of the total diploid DNA (e.g., X, 8, 9, and 13). Interindividual DNA content variation with flow fluorometry was too substantial, however, to confidently diagnose trisomy 21. Fluorescent stains which exclude (variant) simple sequence DNA might overcome this limitation.

Aneuploidy↗