Fluorometry of alpha-amylase.
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Anemia, Sickle Cell↗
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Static and kinetic studies by fluorometry on the interaction between gluconolactone and glucoamylase from Rh. niveus.
A transition-state analog, gluconolactone, was found to partially quench the protein fluorescence of glucoamylase [EC 3.2.1.3] from Rhizopus niveus. The interaction between gluconolactone and the enzyme was studied statically and kinetically at pH 4.5 in terms of fluorescence change. The dissociation constant Kd of the enzyme-analog complex determined by fluorometric titration at 25 degrees (Kd = 1.6 mM) was in good agreement with that obtained by difference spectrophotometric titration (Ohnishi, M. et al. (1975) J. Biochem. 77, 695-703) and with the inhibitor constant determined for the hydrolysis of maltodextrin (Ohnishi, M. et al. (1976) J. Biochem. 79, 1007-1012). The kinetics of the interaction were studied by the fluorescence stopped-flow method. The dependence of the apparent first-order rate constant, kapp, on gluconolactone concentration showed a saturation curve, consistent with a two-step mechanism involving a rapid bimolecular association followed by a slow unimolecular isomerization process. The dissociation constant, KI, for the rapid bimolecular process and the forward and backward rate constants for the isomerization were obtained at 25 degrees and 5 degrees, and the activation parameters were evaluated. It was found that the isomerization process, but not the bimolecular association, is accompanied by fluorescence intensity change, indicating that the former process involves a micro-environmental change of a tryptophan residue(s) of the enzyme. Maltose was found to decrease the rate of interaction of gluconolactone with the enzyme by competing with the analog for the active site.
Binding Sites↗
Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.
Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.
Chelating Agents↗
[Study of molecular mechanisms of muscle contraction using polarization fluorometry].
The review summarizes results of studies on the conformational changes in contractile proteins during muscle contraction. The studies were carried out by polarized fluorescence technique in the UV and visible light. The revealed were alterations of actin and myosin in muscle fiber, taking place at various stages of contractile cycle. Transition from a weak binding state of actomyosin to a strong one was accompanied by F-actin subunit rearrangements, with C- and N-terminals moving relative to the core of thin filament. Myosin light chains and 20-kDa domain of myosin head moved in the same direction as C- and N-terminal regions of actin. The flexibility of actin filaments increased, whereas that of C- and N-terminal regions decreased sharply. Actin-myosin interaction changed dramatically tropomyosin flexibility and caused displacement of the protein relative to C- and N-terminals of actin. Actin structure "freezing" by glutaraldehyde or phalloidin, actin cleavage by subtilisin, as well as actin alteration in denervational atrophy inhibited markedly the intramolecular movement and isometric tension of muscle contraction. Besides, troponin-, caldesmon-, calponin-, and myosin-systems, regulating muscle contraction, modified actomyosin rearrangements in a Ca(2+)-dependent manner. The role of the movement of polypeptide chains in contractile proteins during muscle contraction is discussed.
Actins↗
Fluorometric quantification of low-dose fluorescein delivery to predict amputation site healing.
This retrospective study evaluated quantification of skin fluorescein delivery by fiberoptic fluorometry as a means of predicting the healing potential of an amputation site. Fluorometry uses a dual-channel fiberoptic light guide--one channel transmits blue light to excite the fluorescein in the skin under study, and the other transmits emitted fluorescence from the skin to a photomultiplier tube where it is measured. Ten minutes after intravenous administration of sodium fluorescein (4 to 8 mg/kg), fluorometric readings were obtained at more than 100 reading sites. In the 86 cases without preoperative cellulitis at the site of amputation, preoperative fluorometry clearly distinguished between healing and nonhealing sites. Healing sites averaged 79% of the fluorescence of a healthy reference area (dye fluorescence index [DFI] = 79), while failing sites averaged only 27% (p less than 0.01 by ANOVA). In all but one case where the DFI was greater than 42, the amputation healed. In all cases where the DFI was less than 38, the amputation failed. In general, uncertainty was limited to sites with values between these limits. The technique maintained its high accuracy in patients with diabetes and for distal amputations. However, it was not accurate at sites of active cellulitis (12 cases). There were no significant adverse effects from the slow injection of the low dose of fluorescein used for this technique. We conclude that fluorometry is an effective means of predicting healing in patients undergoing amputation.
Amputation, Surgical↗
Rapid fluorometric assay of bilirubin and bilirubin binding capacity in blood of jaundiced neonates: comparisons with other methods.
The concentrations of total blood bilirubin, albumin-bound bilirubin, and the reserve and total bilirubin binding capacities of 35 neonatal blood samples (28 patients) were determined by automated front-face fluorometry ((hematofluorometer). These values were compared to results of diazo determinations, Sephadex gel filtration, and peroxidase-oxidation methods. Total blood bilirubin level by fluorometry agreed well with the total plasma bilirubin level by diazotization (r = .96, sigma = 1.7 mg/100 ml). Albumin-bound bilirubin concentrations by fluorometry also correlated well with diazo values (r = .95, sigma = 1.9 mg/100 ml) and were slightly lower than the total blood bilirubin concentrations. Values for total bilirubin binding capacity determined by fluorometry agreed well with results obtained for the same specimens by Sephadex gel filtration (n = 28, r = .97, sigma = 1.8 mg/100 ml) and by peroxidase-catalyzed oxidation (n = 25, r = .97, sigma = 1.7 mg/100 ml). The agreement among the results obtained by the three methods indicates a well-defined in vitro end point at which available primary or "tight" binding sites on albumin are saturated with bilirubin. In this clinical experience the coefficient of variation of results with the hematofluorometer was 8.4% for total blood bilirubin and 6.5% for total binding capacity. A comparison of "sick" with "well" infants revealed that the fraction of bilirubin not bound to albumin was significantly different for these two groups. The assays made with the hematofluorometer are quick (10 to 15 minutes) and require only a small quantity (approximately 150 microliters) of blood.
Bilirubin↗
Blood-retinal barrier permeability and its relation to progression of retinopathy in patients with type 2 diabetes. A four-year follow-up study.
Forty patients with late-onset diabetes (age at diagnosis 30 years or more) and minimal retinopathy as found by fundus photography were followed prospectively by repeated examination (baseline, 1 year, and 4 years). The study shows that early retinopathy changes are not permanent or invariably progressive. In the 1st year of follow-up microaneurysms worsened in 25%, improved in 10%, and remained stabilized in 65%. Vitreous fluorometry was able to detect an overall increase of 0.84 +/- 1.06 x 10(-6) min-1 in blood-retinal barrier (BRB) penetration ratios. After 4 years, 16 of the 40 patients had undergone photocoagulation (focal photo-coagulation in 11 and pan retinal photocoagulation in 5). The eyes that needed photocoagulation were the eyes that had higher fluorometry penetration ratios at the patient's entry into the study and showed a higher rate of deterioration during the 1st year of the study (5.54 +/- 1.97 vs 3.11 +/- 1.22 x 10(-6) min-1, P < 0.001, initial values; 1.52 +/- 0.76 vs 0.45 +/- 0.99 x 10(-6) min-1, P < 0.001, annual increase in leakage). The eyes that did not need photocoagulation, 24 out of 40, showed stable fluorometry readings within the 4-year period of follow-up (+0.02 +/- 0.98 10(-6) min-1). Abnormally high vitreous fluorometry values and their rapid increase over time appear to be good indicators of rapid progression and worsening of the retinopathy.
Adult↗
Determination of quinidine in serum by spectrofluorometry, liquid chromatography and fluorescence scanning thin-layer chromatography.
Quinidine is determined in serum by direct and extraction spectrofluorometry, by reflectance fluorescence scanning thin-layer chromatography (TLC), and by high-performance liquid chromatography (HPLC). Least-squares analyses of patients' sera (n = 62) analyzed first by direct fluorometry (x) and then HPLC (y) gave a slope of 0.52, an y-intercept of -0.40, a standard error of estimate of 0.65, and a correlation coefficient of 0.83. Comparison of patients' sera (n = 59) determined by extraction fluorometry (x) and then HPLC (y) gave a slope of 0.998, an y-intercept of -0.175, a standard error of estimate of 0.30, and a correlation coefficient of 0.96. Comparison of patients' sera (n = 36) by HPLC (x) and then reflectance fluorescence scanning TLC (y) gave a slope of 0.837, an y-intercept of 0.152, and a correlation coefficient of 0.94. Methaqualone and oxazepam interfere with HPLC. Within-run precision is 1.6, 1.0, 5.2 and 3.0% by direct fluorometry, extraction fluorometry, TLC and HPLC while between-run precision is 5, 3.5, 9 and 6.0%, respectively.
Chromatography, High Pressure Liquid↗
Fluorescence spectroscopy and imaging of myocardial apoptosis.
Fluorometry is used to detect intrinsic flavoprotein (FP) and nicotinamide adenine dinucleotide (NADH) signals in an open-chest rabbit model of myocardial ischemia-reperfusion injury. Myocyte apoptosis has been shown clinically to contribute to infarct size following reperfusion of ischemic myocardium. A noninvasive means of assessing apoptosis in this setting would aid in the treatment of subsequent ventricular remodeling. We show that in vivo fluorometry can be useful in apoptosis detection in open-chest surgeries. Specific changes in myocardial redox states have been shown to indicate the presence of apoptosis. Two main mitochondrial intrinsic fluorophores, NADH and FP signals, were measured during normoxia, ischemia, and reperfusion experimental protocol. Ischemia was induced by occlusion of the largest branch of the circumflex coronary artery and fluorescence signals are collected by applying two different fluorescence techniques: in vivo fluorometry and postmortem cryoimaging. The first technique was employed to detect FP and NADH signals in vivo and the latter technique uses freeze trapping and low-temperature fluorescence imaging. The heart is snap frozen while still in the chest cavity to make a "snapshot" of the metabolic state of the tissue. After freezing, the ischemic area and its surrounding border zone were excised and the sample was embedded in a frozen buffer for cryoscanning. These two data sets, in vivo fluorometry and low-temperature redox scanning, show consistent extreme oxidation of the mitochondrial redox states (higher redox ratio) suggesting the initiation of apoptosis following reperfusion. This represents the first attempt to assess myocyte apoptosis in the beating heart.
Animals↗
Comparison of results for quantitative determination of morphine by radioimmunoassay, enzyme immunoassay, and spectrofluorometry.
The quantitative results (accuracy and precision) for determination of opiates by radioimmunoassay (RIA), enzyme immunoassay (EMIT), and spectrofluorometry on split samples are compared. A variety of physiological samples were studied, including random urine from a methadone maintenance clinic and postmortem urine, blood, bile, brain, and lung tissue from heroin-induced or heroin-related deaths. The opiate concentrations detected by the two immunoassay methods were in good agreement with each other in the absence of interfering substances which are believed to react with the antimorphine antibodies. The immunoassay results were in agreement within the relative standard deviation with the fluorometry results in 55% of the urine samples and 80% of the blood samples. The immunological methods are superior to fluorometry for quantitation of morphine in urine samples due to quenching interferences in fluorometry from urine. They were comparable to fluorometry for quantitation of morphine in blood samples.
Adult↗
Comparison of blood flow and myoelectric measurements in two chronic models of mesenteric ligation.
OBJECTIVE: To determine whether the predictive accuracy of intestinal motility and blood flow measurements is altered by the magnitude of ischemic damage. DESIGN: Inception cohort study (dogs). Motility was measured using a probe that quantifies both the electromyographic (EMG) measurements and the magnitude of evoked contractile response (ECR). Intestinal blood flow was assessed using Doppler ultrasonography in the marginal artery and perfusion fluorometry, which quantifies fluorescein in the bowel wall in dye fluorescence units. SETTING: Vivarium animal research facilities at a medical school. INTERVENTIONS: The blood supply of a 40-cm length of ileum was ligated in 102 dogs: 52 in which the marginal artery was ligated at two points 8 cm apart (severe model), and 50 in which the marginal artery was ligated only once (moderate model). Twenty-four hours after ligation, the motility and blood flow parameters were measured in normal bowel and at 2-cm intervals within the 40-cm ischemic segment. Resection and anastomosis of ischemic bowel was then performed using either EMG, ECR, or fluorometry to determine the site of resection. OUTCOME MEASURE: Anastomotic leak from progressive ischemia. RESULTS: There were 26 fatal anastomotic leaks, all due to necrosis at the anastomosis. Perfusion fluorometry and ECR measurements did not correlate with survival in either model. An audible Doppler pulse in the marginal artery correlated with survival in the moderate (P < or = .02) but not the severe model (P = .59). The EMG measurements were significantly greater in survivors vs nonsurvivors in both models. CONCLUSIONS: The EMG measurements may be useful in bowel viability assessment. Correlation of Doppler ultrasonographic findings with survival in the moderate model suggests that blood flow measurements may be more reliable in predicting viability in less ischemic bowel.
Anastomosis, Surgical↗
Quantification of fluorescein distribution to strangulated rat ileum.
Following various periods of strangulation, the fates of intestinal segments were predicted by standard clinical criteria and visual (Wood's lamp) and fluorometric (perfusion fluorometer) assessment of fluorescein distribution. With fluorometry, a means of quantifying fluorescence transmitted via a fiberoptic light guide, the delivery and removal of fluorescein were monitored and analyzed. If either was restricted significantly, tissue death was predicted. Analysis of computerized graphic patterns or simple interpretation of fluorometric readings at two time points predicted tissue fate with 98% accuracy and a 93% negative predictive value. Wood's lamp evaluation had only a 53% accuracy and a 33% negative predictive value, while standard clinical criteria had an 81% accuracy and a 53% negative predictive value. Fluorescein leakage in segments which suffered significant endothelial damage provided staining patterns that incorrectly suggested viability. By monitoring elimination as well as uptake of dye, fluorometry provided much greater discrimination than did Wood's lamp inspection in this setting. In addition, fluorometry was readily repeatable within minutes, as fluorescence remaining from a previous injection could be subtracted from new, postinjection values.
Animals↗
Measurement of histamine: a quality control study.
This study evaluated the ability of various laboratories to accurately quantitate histamine in samples of plasma and buffered saline that contained known amounts of histamine. Histamine was dissolved in buffered saline and in plasma and these solutions were lyophilized glass ampules. Sealed ampules were sent to laboratories for analysis of their histamine content by one or more of the following methods: (1) the double-isotope dilution enzymatic method, (2) single-isotope enzymatic method, and (3) manual and automated fluorometry. We analyzed the same solutions in our laboratory by the double-isotope dilution enzymatic method over a 6-mo period; we found a coefficient of variation averaging 26% for these samples over that period of time and our values agreed with theoretical values within 12%. Twelve laboratories analyzed histamine by the double-isotope dilution enzymatic assay. The results revealed a marked variation among laboratories both for the determination of histamine in buffer and, more strikingly, for the determination of histamine in plasma. Three laboratories determined histamine by the single-isotope dilution enzymatic method and one reported results rather close to the standard, while the others reported results that were clearly different from the standards. Five laboratories measured histamine by automated fluorometry and three by manual fluorometry; again, there was marked variation among the results. Overall, these findings indicate that measurements of histamine by different laboratories vary greatly. Thus, absolute values for histamine in biologic specimens in the literature must be regarded with caution.
Chemical Phenomena↗
Comparison of ischemic and reperfusion injury in canine bowel viability assessment.
The purpose of these experiments was to evaluate two methods of bowel viability assessment in two distinct models of intestinal ischemia. Bowel viability was assessed in 32 dogs by means of three methods: (1) a probe that quantified the intestinal electromyographic (EMG) measurements in millivolts (mV), (2) Doppler ultrasonography, and (3) perfusion fluorometry, which quantified serosal blood flow in indexed dye fluorescence units (dfi). Ischemia was created using one of two methods: (1) a chronic model in which the blood supply to 40 cm of ileum was ligated and viability assessed 24 hours later, or (2) an acute model in which the main superior mesenteric artery was occluded for 3 1/2 hours and then released. Viability parameters were assessed every 5 minutes for 30 minutes after release. After viability assessment was completed, the ischemic bowel was resected and anastomosed at the site where the EMG measurements approximated 50% of the values obtained in normal bowel. In the chronic group 3 of 20 dogs died of necrosis in contrast to none of 12 dogs in the acute reperfusion group. In the acute model EMG values steadily increased after reperfusion, stabilizing by 15 minutes after release. Mean EMG values at 15 through 30 minutes after release were significantly greater than the 5- and 10-minute postrelease and prerelease values, suggesting that the electromyogram is affected by reperfusion. Conversely, postrelease fluorometry measurements rapidly increased to levels that exceeded measurements obtained in normal bowel. There was a significant difference in the number of audible Doppler signals in the marginal artery of survivors of the acute vs. the chronic model. Fluorometry measurements in survivors of the acute model (99+/-9 dfi) were significantly greater than measurements in the chronic model (54+/-4 dfi, P<or=0.004). Conversely, intermodel differences in the EMG measurements were not significant. These results show significant differences in the magnitude of ischemic damage induced by reperfusion vs. mesenteric ligation, which had a significant impact on the objective blood flow measurements that were used to predict bowel viability. The results also suggest that intestinal reperfusion injury in dogs has a negligible impact on bowel survival.
Animals↗
Identifying sources of fecal contamination inexpensively with targeted sampling and bacterial source tracking.
Most bacterial source tracking (BST) methods are too expensive for most communities to afford. We developed targeted sampling as a prelude to BST to reduce these costs. We combined targeted sampling with three inexpensive BST methods, Enterococcus speciation, detection of the esp gene, and fluorometry, to confirm the sources of fecal contamination to beaches on Georgia's Jekyll and Sea Islands during calm and stormy weather conditions. For Jekyll Island, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. For the Sea Island beach during calm conditions, the most likely sources of fecal contamination were leaking sewer lines and wildlife feces. The leaking sewer lines were confirmed with fluorometry and detection of the esp gene. For the Sea Island beach during stormflow conditions, the most likely sources of fecal contamination were wildlife feces and runoff discharging from two county-maintained pipes. For the pipes, the most likely source of contamination was bird feces because the percentage of Ent. faecalis was high (30%) and the esp gene was not detected. Sediments were also a reservoir of fecal enterococci for both Jekyll and Sea Islands. Combining targeted sampling with two or more BST methods identified sources of fecal contamination quickly, easily, and inexpensively. This combination was the first time targeted sampling was conducted during stormy conditions, and the first time targeted sampling was combined with enterococcal speciation, detection of the esp gene, and fluorometry.
Animals↗
Serial fluorometric documentation of fluorescein dye delivery.
Since its introduction in 1980, fiberoptic fluorometry has improved the precision and objectivity of the fluorescein test as a means to delineate nutritive blood flow and predict tissue viability. This study was undertaken primarily to determine if fiberoptic fluorometry could be repeated reliably at close time intervals. Four injections of fluorescein dye were administered at varying dose and time schedules to determine what factors influenced the repeatability of this technique. The results confirm the high accuracy of fluorometric indices as a means to predict the ultimate fate of a skin flap. A method of scanning the flap within 2 minutes after injection of 1.5 mg/kg of fluorescein dye correctly predicted the fate of almost 100% of flap sections. Repeated injections of 1.5, 3.0, and 6.0 mg/kg of dye at 15-minute intervals provided virtually identical predictions of flap viability. Thus fluorometry is readily repeatable and should be of value in assessing the state of tissue perfusion as well as changes in perfusion caused by pharmacologic manipulation or surgical intervention.
Animals↗
Lens autofluorescence is increased in newly diagnosed patients with NIDDM.
Lens and cornea autofluorescence has been shown to be increased in patients with insulin-dependent diabetes mellitus and to be positively correlated to glycaemic control and duration of diabetes. We have studied lens and cornea autofluorescence at the clinical onset of non-insulin-dependent diabetes mellitus (NIDDM), in comparison with age-matched subjects with normal glucose tolerance. Fourteen subjects with NIDDM diagnosed less than 6 months prior to the examination were characterised by ocular fluorometry, glycosylated hemoglobin A1c, plasma lipid status, arterial blood pressure, and an oral glucose tolerance test (OGTT). Eleven age- and gender-matched healthy subjects without a family history of diabetes and with a normal glucose tolerance underwent the same examinations. In 11 of the 14 diabetic patients lens autofluorescence was increased to levels higher than the age-related mean + 2SD of healthy subjects. For the entire study population, control and diabetic subjects, lens fluorescence was positively correlated with HbA1c (p < 0.0001, r = 0.73), fasting plasma glucose (p = 0.002, r = 0.60) and the plasma glucose level 2 h after an OGTT (p = 0.004, r = 0.55). Cornea autofluorescence was also significantly increased in the group of newly diagnosed NIDDM patients, but only 9 patients had values above the mean + 2SD of the healthy subjects. NIDDM could be detected by ocular fluorometry with a sensitivity of 79% and a specificity of 100%. We conclude that lens and cornea autofluorescence is abnormally increased in the majority of patients with newly diagnosed NIDDM. The sensitivity and specificity of the method indicate that lens fluorometry may potentially be useful for screening for undiagnosed NIDDM in the general population. Additionally, we propose that the method may be a clinically useful indicator of cumulative glycaemia and risk of development of secondary complications in patients with diabetes.
Adult↗
Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparisons with 32P-postlabeling and ELISA.
A new fluorometric assay was validated for quantification of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in white blood cells (WBC) from humans exposed to polycyclic aromatic hydrocarbons (PAH). This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene derived from acid hydrolysis of BPDE-DNA, and can measure 1 BPDE adduct per 10(8) unmodified nucleotides. The quantity of WBC DNA required depends on the modification level and varies between 5 and 500 micrograms. The assay was applied to seven WBC DNA samples from lung cancer patients, six of whom were heavy smokers, and to three WBC DNA samples from healthy subjects employed in an aluminum production plant. High levels of BPDE-DNA adducts, ranging from 62 to 533 adducts/10(8) nucleotides were found in six out of seven DNA samples from the lung cancer patients. In WBC DNA from healthy persons BPDE-DNA adducts were detected only in two non-smokers, but at a much lower level than in lung cancer patients (4-10 adducts/10(8) nucleotides). Using coded WBC DNA samples, BPDE-DNA adduct levels measured by fluorometry of the B[a]P-tetrols, were compared with the results obtained by 32P-postlabeling (nuclease P1 enrichment) and ELISA measurements. A good correlation and proportionality was found between the levels of BPDE-DNA adducts measured by fluorometry and 32P-postlabeling (r = 0.95, P < 0.001, n = 8). The correlation between fluorometry and ELISA was much lower and not significant (r = 0.61, P = 0.1, n = 6). Moreover, the ELISA grossly overestimated BPDE-DNA adduct levels measured by the other two methods. The results demonstrate that the highly sensitive and specific fluorometric assay is suitable for measuring BPDE-DNA adducts in WBC from humans exposed to benzo[a]pyrene.
Benzo(a)pyrene↗