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PubMed1984

Assessment of neovascularization and timing of flap division.

These studies were undertaken to determine if perfusion fluorometry can provide an accurate, reliable means of monitoring neovascularization and predicting optimal time of flap division. An animal model was developed that permitted daily assessment of neovascularization of the flap using fluorescein injections and the fluorometer. An increase in fluorescence of the pedicle-occluded flap of approximately 12 percent the normal skin fluorescence was associated with 100 percent flap survival. This occurred after only 4 days in the rat. Fluorometry proved valuable in the evaluation of clinical flaps. The fluorescence of flaps during pedicle occlusion was observed to increase with each successive fluorometric evaluation performed at 3- to 7-day intervals. It appears that flaps after pedicle occlusion exhibiting at least 25 percent of the fluorescence of normal skin will tolerate pedicle division. Based on the neovascularization studies of the clinical flaps, all pedicles were severed earlier than originally anticipated. Two cross-leg flaps were successfully divided after 11 days. Quantitative assessment of serial fluorescein injections allows reliable evaluation of neovascularization in clinical interpolation flaps and is useful in the timing of flap division.

Animals↗
PubMed1995

Effects of proinflammatory cytokines on cultivated primary human hepatocytes. Fluorometric measurement of intercellular adhesion molecule-1 and human leukocyte antigen-A, -B, -C, and -DR expression.

Expression of adhesion molecules and human leukocyte antigens on the surface of hepatocytes (HC) may play an important role in the immune reaction in different types of infectious and noninfectious hepatitis, liver graft rejection, and autoimmune liver diseases. The aim of this study was to evaluate the influence of the proinflammatory cytokines IFN-alpha, IFN-gamma, and IL-1 alpha on the expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-A, -B, -C, and -DR on highly purified primary human HC in cell culture. Expression was assessed by semiquantitative measurement of HC in cell culture by means of computer-aided fluorometry after immunofluorescent labeling. Avidin-biotin-immunoperoxidase staining was applied on parallel cultures to evaluate cell purity (> 99%) and to confirm the results obtained by fluorometry. ICAM-1 was expressed constitutively on untreated HC in vitro. Stimulation of HC with IFN-gamma and IL-1 alpha for 24 hr resulted in an increase of ICAM-1 expression. Cultured HC were moderately HLA-A, -B, and -C positive, but HLA-DR negative. Stimulation of HC with 500 U/ml IFN-gamma for 72 hr resulted in an increase of HLA-A, -B, -C, and -DR expression, whereas stimulation with 10 U/ml IL-1 alpha for 72 hr had no influence. By using 5000 U/ml IFN-alpha for 72 hr, we achieved an increase of HLA-A, -B, and -C expression; effects on the other tested antigens were not significant. In contrast to endothelial cells and transformed human hepatocytic cell lines, ICAM-1 on HC was changed more intensively by IFN-gamma than by IL-1 alpha. Furthermore, the results reveal differences in HLA and ICAM-1 expression between HC in vivo and in vitro.

Cells, Cultured↗
PubMed1995

Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig.

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

Intracellular generation of reactive oxygen species during nonhypoxic lung ischemia.

Surface fluorometry with 40 microM hydroethidine (HE) as a probe was used to detect oxidant generation in isolated, ventilated rat lungs during lung ischemia. Ethidium fluorescence due to HE oxidation was continuously monitored with 470 nm excitation and 610 nm emission. Fluorescence increased with ischemia in O2-ventilated lungs [0.98 +/- 0.08 arbitrary fluorescence units (AFU)/min vs. 0.58 +/- 0.07 with control perfusion]. HE oxidation during ischemia was prevented by N2 ventilation but was unaltered by preperfusion with superoxide dismutase. Ethidium fluorescence in homogenate prepared from lungs subjected to 1 h of nonhypoxic ischemia was increased (16.8 +/- 1.5 vs. 9.8 +/- 0.4 AFU/mg protein in control) but was unchanged in lungs that had been N2 ventilated. Microfluorographs of HE perfused and fixed lung sections demonstrated marked generalized increases in ethidium fluorescence with ischemia compared with control perfusion. Ischemia resulted in significant increases in tissue thiobarbituric acid reactive substance (176 +/- 13 vs. 44 +/- 3 pmol/mg protein for control) and in lung conjugated dienes (0.90 +/- 0.07 vs. 0.48 +/- 0.06 U/mg protein for control), indicating peroxidation of lung lipids. These results indicate that lung ischemia leads to intracellular oxidant generation that can be continuously monitored by surface fluorometry.

Animals↗
PubMed1993

Distribution of autofluorescence in the rabbit corneal epithelium.

Autofluorescence from reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) was measured in order to detect the difference in redox states in rabbit corneal epithelium. The enucleated rabbit eye was mounted in an eye bank eye container with McCarey-Kaufman medium, and the autofluorescence was measured using ocular redox fluorometry as a function of depth. The PN signal distributed evenly whereas the Fp signal was greater in the posterior epithelial region than in the anterior region (p < 0.05). The PN/Fp ratio, a sensitive indicator of tissue redox state, was less in the posterior region. After the application of 1 mM of potassium cyanide in the medium, the ratio increased significantly in each layer (p < 0.001), and the difference between anterior and posterior region diminished. These results indicate that ocular redox fluorometry has the potential to resolve the redox states of the various layers of the corneal epithelium. The posterior region of the epithelium is more active in mitochondrial respiration than the anterior region.

Animals↗
PubMedDate not supplied

An improved fluorometric assay of rat serum and plasma converting enzyme.

The most sensitive nonradiometric routine assay for angiotensin-converting enzyme (ACE) activity uses fluorometry to detect His-Leu released from Hip-His-Leu. Our results indicate that, in contrast to human serum, rat serum and plasma contain large and variable amounts of dipeptidase activity that lead to a subestimation of the ACE activity measured in 0.1 M potassium phosphate buffer, pH 8.3, containing 0.3 M NaCl, the most commonly used assay for human serum and tissue ACE. We describe and validate an assay for 1 to 10 microL rat and human serum or plasma using 5 mM Hip-His-Leu in 500 microL of 0.4 M sodium borate buffer, pH 8.3, containing 0.9 M NaC1 at 37 degrees C that reduced the subestimation error to less than or equal to 3% (rat serum) and less than or equal to 0.1% (human serum) and increased the ACE activity twofold to threefold. The Km and Vmax are reported for rat serum ACE (Hip-His-Leu) and dipeptidase (His-Leu) in borate buffer and phosphate buffer. Rat serum ACE hydrolysis of Hip-His-Leu measured by fluorometry correlated (r = 0.99, p less than 0.05) with the hydrolysis of angiotensin I measured by high-performance liquid chromatography. A direct method based on amino acid analysis is described for evaluating the dipeptidase error of complex mixtures such as tissue extracts and other physiological fluids. We have found that the assay can be used to measure ACE activity in 25 samples (in duplicate) in 2 hours with small intraassay (2.2%) and interassay (3.9%) coefficients of variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗
PubMed1998

[Highly effective separation and highly sensitive detection for clinical chemistry and biochemistry].

Highly effective separation and highly sensitive detection reagents for clinical chemistry and biochemistry were developed and their applications were investigated. The sensitive detection of carboxylic acids was accomplished using 9-anthryldiazomethane (ADAM) which gave intensely fluorescent derivatives from carboxylic acids without catalysts or heating. 1-Pyrenyldiazomethane was then synthesized and proved to react also readily with carboxylic acid and more sensitive than ADAM. The optical resolution of amino acid enantiomers was achieved using 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). GITC derivatized enantiomeric amino acids under mild conditions to give highly hydrophobic diastereomers which could be resolved on conventional reversed phase columns and easily detected by the absorption based on the thiourea structure. Then we devised an o-phthalaldehyde-N-acetylcystein reagent (OPA/NAcCys) giving diastereomers which were also resolved on a reversed phase column and detected fluorometrically with excellent sensitivity. OPA/NAcCys was useful for the assay of D-amino acids in the blood of uremic patients. The hypochlorite-thiamine method for the assay of proteins and peptides was established providing sensitive fluorometry, which well reflected the number of peptide groups in the molecule. This principle was applied to the assay using N-chlorodansylamide, designed for the fluorometry of peptides. The alkaline ninhydrin method was applied to the detection of guanidino compounds in the blood of uremic patients. Several fluorometric methods for the simultaneous detection of reducing and non-reducing carbohydrates, and guanidines were found to be useful reagents for this purpose because these compounds were resistant to the oxidation with periodate. Then protamine-bound columns were prepared for the separation of carbohydrates on HPLC, which showed excellent recovery of reducing carbohydrates in comparison with conventional alkylamino columns.

Amino Acids↗
PubMed2006

Biocompatible nanometal particle fluorescence enhancers.

Fluorometry has been used for more than a century to enhance the understanding of phenomena in molecular biology. With recent advances in light detection techniques, it is even more actively used in research now. The effectiveness of fluorometry highly depends on the intensity of the fluorescence from the target molecules in contrast to the background. Instrumentation for amplifying fluorescence signals may not selectively enhance signals only from the target. Therefore, a method to retrieve more fluorescence emission from a fluorophore is beneficial. Nanometal particles have high-density surface plasmon polariton fields, and when a fluorophore is placed within these fields, the electrons involved in the fluorescence emission may be altered in their energy states. The level of alteration depends upon the metal type, size of the particle, quantum yield of a fluorophore, and, most importantly, the distance between the nanoparticle and the fluorophore. Some organic solvents are also found to enhance fluorescence, possibly by the dipole-dipole coupling between the fluorophore and the solvent, resulting in an increase in the energy gap between the excited and the ground states. In this article, study results of the application of nanometal particles and biocompatible solvents for increasing the sensitivity/resolution in fluorophore-mediated biosensing/bioimaging are reviewed.

Biosensing Techniques↗
PubMed2000

Concentrations of elastinolytic metalloproteinases in respiratory tract secretions of healthy horses and horses with chronic obstructive pulmonary disease.

OBJECTIVES: To determine whether samples of tracheal epithelial lining fluid (TELF) obtained from horses have elastinolytic activity characteristic of metalloproteinases, to compare elastinolytic activity in TELF obtained from healthy horses and horses with chronic obstructive pulmonary disease (COPD), and to determine whether chemically modified tetracycline-3 (CMT-3) inhibits elastinolytic activity in TELF ANIMALS: 10 horses with COPD and 10 healthy control horses. PROCEDURE: Zymography and fluorometry were used to measure elastinolytic activity, and EDTA was used to inhibit elastinolytic activity and verify that the activity was attributable to metalloproteinases. Possible inhibition of elastinolytic activity with CMT-3 was studied in vitro. RESULTS: Elastinolytic activity was found in TELF obtained from all horses, and this activity was significantly higher in TELF obtained from horses with COPD than in TELF obtained from healthy horses. For all samples, EDTA and CMT-3 inhibited elastinolytic activity. CONCLUSIONS AND CLINICAL RELEVANCE: Elastinolytic activity is detectable in TELF obtained from horses and seems to be attributable to metalloproteinases. Elastinolytic activity in TELF is significantly inhibited by CMT-3. Elastinolytic activity in TELF can be detected by means of zymography or fluorometry. Increased elastinolytic activity may reflect destruction of pulmonary tissue in horses with COPD. Chemically modified tetracyclines such as CMT-3 may provide an additional treatment possibility for horses with COPD.

Animals↗
PubMed1992

Metabolic and morphologic changes in the corneal endothelium. The effects of potassium cyanide, iodoacetamide, and ouabain.

The metabolic pathways of glycolysis and mitochondrial respiration in the corneal endothelial cell are the primary sources of the adenosine triphosphate (ATP) necessary to maintain endothelial structure and pump fluid to maintain the corneal stroma in its normally dehydrated and transparent state. The correlation between endothelial metabolism and morphology in rabbits was studied for 7 days after the application of three different agents: (1) iodoacetamide, used to inhibit ATP synthesis from both glycolysis and respiration; (2) potassium cyanide (KCN), used to inhibit ATP synthesis from respiration only; and (3) ouabain, used to inhibit fluid pumping but not ATP synthesis. After application of each of these three drugs to the corneal endothelium, changes in endothelial morphology were measured. The greatest change resulted from the use of iodoacetamide. Specular microscopic examination of the endothelium after the application of iodoacetamide showed progressive degradation of the integrity of the cellular structure; after 6 hr, there were no discernible cell borders. In those corneas treated with either KCN or ouabain, only minor changes in the endothelium were seen during the full 7 days of the investigation. Computer-assisted morphometric analysis showed an increase in the coefficient of variation of both cell area and perimeter in all cases. This increase was greater in the corneas treated with ouabain than those treated with either iodoacetamide or KCN. Redox fluorometry showed that the metabolic ratio (autofluorescence of reduced pyridine nucleotides divided by that of oxidized flavoproteins) decreased significantly in the iodoacetamide-treated corneas, increased significantly in the KCN group, and showed no significant change in the corneas in the ouabain group, all compared with a control group. The results showed that (1) when ATP produced by both glycolysis and respiration was inhibited by 0.1 mmol/l iodoacetamide, the endothelial cells could not survive, but (2) when ATP synthesis produced by respiration alone was inhibited by 1.0 mmol/l KCN, the cells could survive for at least 1 wk on the ATP produced by anaerobic glycolysis. Furthermore, the polymegathism seen after application of ouabain, a drug that is not believed to affect ATP synthesis but inhibits the endothelial pump function, is greater than that seen as a result of reduced pump function caused by inhibited respiration produced by 1.0 mmol/l KCN. Combining specular microscopy, computer-assisted morphometric analysis, redox fluorometry, and corneal pachymetry allowed correlations between corneal endothelial metabolism, pump function, and morphology to be studied.

Animals↗
PubMed1992

[Metabolic analysis of the diseased human corneal endothelium].

Redox states of the corneal endothelium in 42 recipient corneas obtained at the penetrating keratoplasty were measured non-invasively using ocular redox fluorometry. Autofluorescence from reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) were measured, and the PN/Fp ratio was used as an indicator of the redox state. Endothelial damage was graded as normal, mildly damaged, moderately damaged, and severely damaged, based on the histopathological findings. Mildly damaged endothelium showed a significantly higher PN/Fp ratio than the that in normal endothelium whereas the ratio was significantly lower in the severely damaged endothelium. These changes in the redox state may represent compensation and decompensation processes of the endothelial metabolism. Ocular redox fluorometry was shown to be useful for the evaluation of the metabolic state in the human corneal endothelium.

Adult↗
PubMed1991

Transcutaneous determination of tissue dihematoporphyrin ether content. A device to optimize photodynamic therapy.

Photodynamic therapy involves the use of light of appropriate wavelength to excite a photosensitizer resulting in tissue destruction. The photosensitizer dihematoporphyrin ether is selectively retained in tumors allowing for tumor destruction while sparing normal structures. Accessibility of skin tumors makes them well suited for photodynamic therapy. Tissue and tumor dihematoporphyrin ether content is estimated based on the amount of dihematoporphyrin ether administered. In our study, skin dihematoporphyrin ether content was measured in guinea pigs transcutaneously by a hand-held fluorometer and compared with dihematoporphyrin ether determinations done on skin biopsy specimens. Fluorometry was performed on guinea pigs receiving 0, 2.5, 5, 10, and 25 mg/kg of dihematoporphyrin ether. Transcutaneous measurements of skin fluorescence increased with increasing dihematoporphyrin ether dose and correlated well with skin dihematoporphyrin ether content as determined by extracting dihematoporphyrin ether from skin samples. Transcutaneous fluorescent measurements of guinea pigs given 0 and 2.5, 2.5 and 5, 5 and 10, and 10 and 25 mg/kg of dihematoporphyrin ether differed in a statistically significant manner. Transcutaneous fluorometric determination of dihematoporphyrin ether content and extraction of dihematoporphyrin ether from skin samples were able to reflect differences in dihematoporphyrin ether dosing and presumably skin dihematoporphyrin ether content. However, transcutaneous fluorometry provides an instantaneous estimate of tissue dihematoporphyrin ether without the need for a tissue sample. This may provide a clinical tool to predict more accurately the optimal light dose necessary to maximize photodynamic therapy.

Animals↗
PubMed1990

Deduction of DNA from artificial aggregates of erythrocyte nucleosomes.

The nucleosomal aggregates were obtained by micrococcal nuclease treatment to chicken erythrocyte nuclei. There still leaves a bulk of nucleosomes. We used two different DNA assay methods to determine DNA in in situ nucleosomes; the Feulgen DNA assay which shows a positive apurinic acid, and the fluorometry with Hoechst 33258 which reveals only intact AT base pairs. On applying those methods to the aggregates, even in a short digestion, Feulgen DNA remains only about 1/4 of the non-digested nuclei and the fluoroassay leaves only a trace amount of AT base pairs. Thus, the nucleosomes derived from the heterochromatin of erythrocytes are not preserved as the residual DNA of Feulgen hydrolysis. This also suggests that the bulk of nucleosomal DNA is masked and sensitive to neither the Feulgen assay nor the fluorometry of AT base pairs.

Animals↗
PubMed1985

Fluorometric prediction of successful amputation level in the ischemic limb.

The present study was undertaken to compare fluorometric documentation of fluorescein dye delivery with the standard means of determining the level at which an amputation should be performed in the dysvascular extremity. Thirty-nine patients underwent lower-extremity amputation at the level determined by the surgeon based upon physical examination, angiography, segmental pressure indices, and/or pulse volume recordings. In addition, fiberoptic fluorometry was performed preoperatively. After intravenous administration of sodium fluorescein (4-8 mg/kg), fluorometric readings were obtained by placing the fluorometer's light guide on 126 reading sites. Fluorometric findings were evaluated retrospectively, and therefore did not influence the surgeon's decision. Of the 39 amputations performed overall, only 26 healed. The accuracy of the standard criteria was lowest for the 20 below-ankle amputations, where only 12 cases healed. Alternatively, fluorometric indices separated healing from nonhealing sites in 36 of the 39 cases and in 18 of the 20 below-ankle amputations. Overall, healing sites averaged 94 percent of the fluorescence of the healthy reference area, while nonhealing sites averaged only 29 percent. We conclude that fluorometry should prove to be a valuable adjunct in the assessment of the dysvascular extremity. It uses a low dose of dye, is easy to perform, and is readily repeatable.

Amputation, Surgical↗
PubMed1995

Changes of corneal redox state in diabetic animal models.

Metabolic and biochemical changes of the corneal epithelium and endothelium were studied in experimental diabetic animal models. Ocular redox fluorometry was used to noninvasively determine tissue reduction-oxidation (redox) changes in organ cultured rabbit corneas incubated in high glucose concentration media, alloxan-induced diabetic rabbits, and nonobese diabetic mice. The ratio of autofluorescence from reduced pyridine nucleotide to oxidized flavoproteins (PN/Fp) was used as the indicator of the redox state. Chemical assays for NADH and NAD+ were performed on in vitro materials. Analysis of corneal endothelial morphology using specular microscopy was performed to study possible correlations with metabolic changes. Both the PN/Fp and NADH/NAD+ ratios increased in the corneal endothelium under all experimental conditions. Changes in redox state were not observed in the corneal epithelium in any of the models. Morphologic analysis of the corneal endothelium revealed no significant changes. These results indicate that redox changes occur in the diabetic corneal endothelium, but not the corneal epithelium. Ocular redox fluorometry is capable of detecting changes in the corneal endothelial redox state noninvasively.

Animals↗
PubMed1993

Increased expression of the epidermal growth factor receptor in human epidermal keratinocytes after exposure to ionizing radiation.

The effect of exposure of human epidermal keratinocytes to ionizing radiation, both in vivo and in vitro, on the expression of the epidermal growth factor receptor (EGF-R) was studied on the protein, mRNA, and functional levels. Quantitative fluorometry of short-term organ cultures incubated with a monoclonal antibody against human EGF-R revealed a dose-dependent increase of EGF-R expression 24 h after irradiation with 4 and 6 Gy, with an additional increase after 48 h. In biopsy specimens from patients undergoing radiation therapy a markedly increased expression could be determined by quantitative fluorometry during radiation therapy which wa still considerably above the baseline level 4 weeks after termination of treatment. Radioligand binding assays demonstrated a 50% increase in 125I-EGF binding to primary keratinocytes and A431 cells, at doses of 1 Gy, with a further increase after 72 and 96 h. Northern blots were performed with total RNA from two human epidermal cell lines (SCLII and A431). In A431 cells, increased EGF-R transcript levels could be detected 48 h after irradiation. In cells of the SCLII cell line, EGF-R expression was not affected by irradiation. These results were confirmed by semiquantitative polymerase chain reaction of primary cultured keratinocytes, demonstrating an increase of transcripts of EGF-R 24 h after irradiation with single doses of 6 Gy. Thus exposure to ionizing radiation leads to an increased expression of functionally intact EGF-R in human keratinocytes, at the protein and mRNA levels, both in vitro and in vivo; we hypothesize that this effect is part of a stress program of epidermal cells in response to ionizing radiation, ensuring rapid repopulation of irradiated areas.

Base Sequence↗
PubMed1998

Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours.

We compared the screening interpretation of fluorometric analytical results for phenylketonuria (PKU) with tandem mass spectrometry (MS/MS) in filter paper blood spots collected from newborns <24 h of age. In MS/MS, both Phe and Tyr are quantified. Two hundred and eight blood spots collected from infants <24 h of age were retrieved from storage from the California newborn screening program. These samples had been categorized on the basis of fluorometric analysis as initial negative, initial positive for hyperphenylalaninemia with negative determination on recall, or initial positive for hyperphenylalaninemia and confirmed on follow up as PKU or variant hyperphenylalaninemia. The retrieved samples were analyzed in a blinded fashion using MS/MS. Correlation analysis of fluorometry vs MS/MS for Phe concentration was high, with a Pearson correlation coefficient of 0.817. When 180 micromol/L was used as the cutoff Phe concentration for MS/MS and 258 micromol/L was used as the cutoff for fluorometry, all infants with confirmed classical PKU and variant hyperphenylalaninemia were detected. MS/MS analysis reduced the number of false-positive results from 91 to 3. Simultaneous quantification of Phe and Tyr by MS/MS with the use of a cutoff Phe/Tyr molar ratio of 2.5 further reduced the number of false positives to 1. Samples from affected infants showed a discernible trend of increasing Phe concentration and Phe/Tyr molar ratio with age of collection. These results demonstrate the utility of MS/MS in the routine PKU screening of early-discharge newborns. MS/MS reduces the false-positive rate of fluorometric screening almost 100-fold because of the improved accuracy and precision of Phe measurement and simultaneous confirmation with the Phe/Tyr molar ratio. In addition to the detection of PKU, MS/MS can also detect other aminoacidopathies and disorders of fatty acid and organic acid metabolism with lower false-positive rates than other methods currently used in newborn screening programs.

Blood Specimen Collection↗
PubMed1993

Chicken embryo fibroblasts exposed to weak, time-varying magnetic fields share cell proliferation, adenosine deaminase activity, and membrane characteristics of transformed cells.

Chicken embryo fibroblasts (CEF) exposed to a sinusoidally varying magnetic field (SVMF) (100 Hz, 700 microT, for 24 h) showed a remarkable rise of segmental rotational relaxation rate of adenosine deaminase (ADA, EC 3.5.4.4) as determined by multifrequency phase fluorometry. Pyrene-labeled, small subunit ADA was applied to cultured (normal) CEF, which have available and abundant ADA complexing protein (ADCP) on their plasma membranes. Sine-wave-modulated fluorometry of the pyrene yielded a profile of phase angle vs. modulation frequency. In SVMF-treated cells and in Rous-sarcoma-virus (RSV) transformed cells the differential phase values at low modulation frequencies of the excitation are remarkably reduced. This effect is magnetic rather than thermal, because the temperature was carefully controlled and monitored; nevertheless to further check this matter we studied CEF, infected by the RSV-Ts68 temperature-sensitive mutant (36 degrees C transformed, 41 degrees C "revertant"). When grown at 36 degrees C in the SVMF, cells did not show the slightest trend towards reversion, as would be expected had there been local heating. Concomitant with the increased segmental rotational relaxation rate of ADA, there was a decrease in fluorescence lifetime and a slight, yet significant, increase in membrane lipid "microfluidity." These biophysical observations prompted us to examine the effect of SVMF on cell proliferation and ADA activity (a malignancy marker): higher rates of cell proliferation and reduced specific activity of ADA were observed.

Adenosine Deaminase↗