Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin.

Nanobiotechnological improvements defined on the utilization of biological materials and principles have enormously partaken to revolutionize physical, chemical, and biological sciences. However, the exploration of plant nanobiotechnology is still in its outset. The search for novel tools to monitor plant biomolecules is an emerging issue for the nanobiotechnologists. Given this, a genetically encoded FRET-based nanobiosensor has been developed to monitor the popular plant cardiac glycoside - digoxin, which is used as the most common prescription drug for heart-related illnesses across the world. Digoxin is sourced from the leaves of the foxglove plant (Digitalis purpurea L.) and has a significant demand in the medical sector. Moreover, with the rising popularity of the herbal formulations in the global market, attention towards the authentication and quality control of the herbal drugs is important. Furthermore, digoxin has a very narrow therapeutic range, i.e., 0.6 nM - 2.6 nM. Therefore, strict monitoring of blood digoxin levels is necessary. Besides, previously used techniques for drug authentication and quantification of small-molecule drugs in blood samples are not the best choice available. The nanobiosensor is based on the FRET (Fluorescence Resonance Energy Transfer) mechanism, and it is constructed in such a way that it gives a changed FRET output in the presence of digoxin. Two fluorophores, enhanced cyan fluorescent protein (ECFP) and Venus, were attached on either end of the sensory domain - human nuclear receptor ROR-gamma (RORγt). The developed nanobiosensor was named as fluorescent indicator protein for digoxin, (FLIP-digoxin). The ligand binding affinity of FLIP-digoxin was calculated as 425 µM. Affinity mutants of the FLIP-digoxin were also generated to measure digoxin in wide concentration ranges. This sensor offers high-throughput qualitative analysis of digoxin in Digitalis preparations procured from local drug stores. It confirms the authenticity of the preparations through the detection of digoxin. The FLIP-1n was also able to monitor digoxin concentration in serum samples in lesser than 5 min. The nanobiosensor was found pH stable, digoxin-specific, non- interfered by the biological serum species and can perform high throughput screening of the Digitalis powder, infusion and tincture preparations.

Biotin at High Concentration Interferes with the LOCI Digoxin Assay but the PETINIA Phenytoin Assay Is Not Affected.

Many automated immunoassays incorporate biotinylated antibodies and streptavidin-coated magnetic beads in the assay design. Biotin at elevated concentrations may interfere with these immunoassays. We evaluated potential interference of biotin on serum digoxin (LOCI assay utilizing biotinylated antibody) and phenytoin (PETINIA assay; no biotinylated antibody) measurements using the Vista 1500 analyzer. Aliquots of drug-free serum pool were supplemented with various biotin concentrations (range: 1 ng/mL to 250 ng/mL) followed by measuring apparent digoxin and phenytoin levels using appropriate immunoassays. In the second set of experiments, one serum pool was prepared from patients taking digoxin and another from patients taking phenytoin. Then aliquots of these serum pools were further supplemented with biotin followed by measuring digoxin or phenytoin concentrations. We observed apparent digoxin levels at 50 ng/mL biotin concentration or higher and also significant interference of biotin in serum digoxin measurement at a biotin concentration of 250 ng/mL. In contrast, we observed no interference of biotin in serum phenytoin measurement. We conclude that biotin interferes with the LOCI digoxin assay at a high concentration only.

Clinical judgement perplexed by initially undisclosed use of herbal medicine and unexpected cross-reactivity of immunoassay.

We report a case of symptomatic bradycardia caused by consumption of a Chinese herbal medicine which was initially undisclosed to the attending emergency physician. The scientific name of the herb is Panax japonicus. Electrocardiogram revealed sinus bradycardia. Laboratory tests were normal except for the detection of a high serum digoxin level. Further interrogation of the patient eventually disclosed ingestion of the herb which, however, did not contain any digoxin. Other active ingredients in the herb include various types of ginsenoside. These are digoxin-like substances that had caused the observed false-positive detection of digoxin by fluorescence polarization immunoassay due to cross-reactivity. Our case-report provides an important insight about a blind-spot in the field of laboratory medicine (clinical pathology), namely, the false positive detection of digoxin due to crossreactivity in the immunoassay when we come across digoxin-like substances in clinical scenarios, which has barely received attention in the medical literature. It also conveys a clear educational message that with full understanding of the laboratory methodology and its mechanistic rationale there are actually some tricks-of-the-trade that allow us to optimize the specificity of the biochemical tests and the treatment of digoxin-like substances overdose.

Rapid detection of oleander poisoning by Dimension Vista digoxin assay (Flex Reagent Cartridge).

Oleander poisoning can be detected by digoxin immunoassays and for last two decades the fluorescence polarization immunoassay (FPIA) has been used for rapid detection of oleander poisoning in clinical laboratories. Recently, Abbott Laboratories (Abbott Park, IL) discontinued this assay. Therefore, we explored the possibility of using another digoxin assay (Dimension Vista Flex Reagent Cartridge, Tina Quant, EMIT 2000 and old FPIA assay for comparison) for rapid detection of oleander poisoning. When aliquots of drug-free serum pools were supplemented with pure oleandrin or oleander extract, we observed the highest apparent digoxin values using Dimension Vista digoxin assay (Flex Reagent Cartridge). We also observed significant apparent digoxin values in vivo in sera of mice both 1 and 2 hr after feeding with oleander extract. When a serum pool prepared from patients taking digoxin was further supplemented with various amounts of oleander extract, the highest falsely elevated digoxin values were observed with Dimension Vista digoxin assay. Monitoring free digoxin using Dimension Vista digoxin assay (Flex Reagent Cartridge) did not eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract and such effect can be monitored by observing significant reduction in apparent free digoxin levels in the presence of Digibind as measured in the protein-free ultrafiltrate using Dimension Vista digoxin assay (Flex Reagent Cartridge).

A new enzyme-linked chemiluminescent immunosorbent digoxin assay is virtually free from interference of spironolactone, potassium canrenoate, and their common metabolite canrenone.

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are sometimes used in conjunction with digoxin for patient management. Spironolactone, potassium canrenoate, and their common metabolite canrenone interfere with serum digoxin measurement using various immunoassays. Recently a new enzyme-linked chemiluminescent immunosorbent digoxin assay (ECLIA-Digoxin) became commercially available for application on the ADVIA IMS 800i modular system (Bayer HealthCare, Tarrytown, NY). We investigated the potential interference of spironolactone and related compounds in this assay by comparing the results with the fluorescence polarization immunoassay (FPIA), which is known to have significant cross-reactivity with these compounds as well as a turbidimetric assay for digoxin with no known cross-reactivity with spironolactone and related compounds. Aliquots of drug free serum were supplemented with therapeutic and above therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured. No apparent digoxin concentration was observed using the ECLIA-Digoxin or turbidimetric assay. When serum pools prepared from patients receiving digoxin were further supplemented with these compounds, we observed no significant change in digoxin concentrations in the presence of these compounds with the ECLIA-Digoxin. We conclude that this assay is virtually free from interferences from spironolactone, potassium canrenoate and their common metabolite canrenone.

Rapid detection of oleander poisoning using digoxin immunoassays: comparison of five assays.

Oleander is an ornamental shrub that grows in the United States, Australia, India, Sri Lanka, China, and other parts of the world. All parts of the plant are poisonous because the presence of cardiac glycoside oleandrin. Despite its toxicity, oleander extract is used in folk medicines. Because of its structural similarity, oleandrin cross-reacts with the fluorescence polarization immunoassay (FPIA) for digoxin. We studied the potential of detecting oleandrin in serum using 5 common digoxin immunoassays (FPIA, MEIA, both from Abbott; Beckman digoxin assay on Synchron LX, Chemiluminescent assay, CLIA from Bayer Diagnostics) and a recently FDA-approved turbidimetric assay on the ADVIA 1650 analyzer (Bayer). Aliquots of drug-free and digoxin-like immunoreactive substances (DLIS)-free serum pools were supplemented with ethanol extract of oleander leaves or oleandrin (Sigma Chemicals) in amounts expected in vivo after severe overdose. We observed significant apparent digoxin concentration with FPIA, Beckman, and the new turbidimetric assay (1 mL drug-free serum supplemented with 5.0 microL of oleander extract: apparent digoxin 2.36 ng/mL by the FPIA, 0.32 ng/mL by the MEIA, 0.93 ng/mL by the Beckman, 0.82 ng/mL by the new turbidimetric assay). The CLIA showed no cross-reactivity. Similar observations were made when serum pools were supplemented with oleandrin. Because cross reactivity should be tested in the presence of the primary analyte, we supplemented serum pools prepared from patients receiving digoxin with oleander extract or oleandrin. The measured digoxin concentrations were falsely elevated with the FPIA, Beckman, and turbidimetric assays, the highest false elevation being observed with the FPIA. Surprisingly, apparent digoxin concentrations were falsely lowered when MEIA was used. Digibind neutralizes free apparent digoxin concentration in vitro in serum pools supplemented with oleander extract, and this effect can be measured by the FPIA. We conclude that FPIA is most sensitive to detect the presence of oleander in serum. In contrast, the CLIA (no cross-reactivity) should be used for monitoring digoxin in a patient receiving digoxin and self-medicated with a herbal remedy containing oleander.

[Preparation of biologically transformed raw material from woolly foxglove Digitalis lanata Ehrh and isolation of digoxin from it]. / Poluchenie biotransformirovannogo syr'ia naperstianki sherstistoi Digitalis lanata Ehrh i vydelenie iz nego digoksina.

A biotechnological approach is proposed for conservation of a terraneous part of woolly foxglove under anaerobic conditions with a subsequent air-sun drying of the biologically transformed raw material. During the conservation primary foxglove glycosides completely convert to secondary ones which do not transform further. A simple method is described for preparation from the transformed raw material of an enriched glycoside fraction with the yield of 3.6% and for isolation from this fraction of highly purified digoxin with the yield of 0.06% of the starting raw material, and the other secondary glycosides can be also isolated.

Study on mechanism of action of Chinese medicine Chan Su: dose-dependent biphasic production of nitric oxide in trophoblastic BeWo cells.

BACKGROUND: Chan Su, a traditional Chinese medicine, is used for treating the heart diseases and other systemic illnesses. Our studies with animal model have revealed its role in increasing intracellular calcium concentration in cardiomyocytes. Nitric oxide (NO), a second messenger molecule, and its metabolites have been demonstrated to maintain and modulate multiple physiologic functions including the cardiovascular and reproductive systems. In order to explore the mechanism of action of Chan Su, we studied the ability of Chan Su to stimulate NO production in cultured trophoblastic BeWo cells. MATERIALS AND METHODS: BeWo cell is a cloned established cell line purified from human choriocarcinoma. These cells have some similarities in biological behavior with endothelial cells. Therefore, BeWo cell line may act as a model system for production of nitric oxide by Chan Su both in placenta and in cardiovascular tissue, and the results can easily be extrapolated to cardiomyocytes. Very small amount of ethanol extract of Chan Su was added to the cultured cells in KBM buffer and a chemiluminescence system was used for the measurement of nitric oxide. The amounts of Chan Su extract added to cultured cells were comparable to expected level of Chan Su in human serum after ingestion. We also repeated these experiments with bufalin, the active component of Chan Su. RESULTS: The ethanol extract of Chan Su (5 and 10 microg/ml) significantly increased NO production up to 110% of basal control value, but higher concentrations (40 and 80 microg/ml) of Chan Su (as expected in an overdose) resulted in decreased NO production below the control level. This biphasic effect on nitric oxide production was also observed with bufalin, the active component of Chan Su responsible for its digoxin-like immunoreactivity. The presence of bufalin in Chan Su preparation was confirmed by thin layer chromatography (TLC) analysis. CONCLUSIONS: Chan Su as well as bufalin is able to modulate the production of NO in BeWo cell line.

Effect of the traditional Chinese medicines Chan Su, Lu-Shen-Wan, Dan Shen, and Asian ginseng on serum digoxin measurement by Tina-quant (Roche) and Synchron LX system (Beckman) digoxin immunoassays.

Chan Su, Lu-Shen-Wan, Dan Shen, and Asian ginseng are traditionally used to treat a number of conditions, including cardiovascular disease. All of these traditional Chinese medicines exhibit cardioactive properties. Digoxin is a cardioactive drug with a narrow therapeutic range (0.8-1.9 ng/mL). A patient taking digoxin may also take these Chinese medicines for their cardiotonic effects. Moreover, the active components of these medicines that are responsible for cardiotonic effects bear structural similarities to digoxin. Therefore, we studied the potential interference of these Chinese medicines with two digoxin immunoassays--the Tina-quant (Roche Diagnostics) and the Beckman (Synchron LX system)--and compared the values with the fluorescence polarization immunoassay (FPIA; Abbott Laboratories). When very small amounts (2-5 microL) of aqueous extract of Chan Su or Lu-Shen-Wan were added to drug-free serum, we observed high digoxin-like immunoreactivity with the FPIA. In contrast, when ethyl acetate extract of Dan Shen or microliter amounts of ginseng extract were added to drug-free serum, we observed modest digoxin-like immunoreactivity with the FPIA, but no apparent digoxin activity with the Roche and Beckman digoxin immunoassays. When aliquots of a digoxin pool prepared from patients receiving digoxin were supplemented with these Chinese medicines, we observed the most significant interference with the FPIA. The presence of endogenous digoxin-like immunoreactive substances can have additive effects with these Chinese medicines and falsely increase apparent digoxin levels by the FPIA. On the other hand, the Roche and Beckman assays were free from interference from DLIS but showed significant interference from Chan Su and Lu-Shen-Wan. We conclude that the FPIA showed the most significant interference from all four of the Chinese medicines we studied. However, the Roche and Beckman assays showed no interference from two (Dan Shen and Asian ginseng) of the four Chinese medicines we studied.

Drug-herb interactions: unexpected suppression of free Danshen concentrations by salicylate.

The general population of the U.S. uses over-the-counter herbal medicines. Danshen is a Chinese herbal product used for the treatment of cardiovascular disease. In a previous study we showed that Danshen has significant digoxin-like immunoreactivity, and we used this parameter to monitor total and free Danshen activities in sera (10). In this report we demonstrated strong protein binding of Danshen (50-70%), and we also identified albumin as the major serum protein that binds Danshen. Because salicylate, which is also strongly bound to albumin, is a widely used over-the-counter medicine in the U.S., we studied Danshen-salicylate interaction in vitro. We observed no significant change in free Danshen concentrations as measured by free-digoxin-like activity when salicylate concentrations were subtherapeutic (< or = 100 microg/mL). With therapeutic concentrations of salicylate (> or = 150 microg/mL), the free Danshen concentrations significantly decreased from the control. On the other hand, Danshen can displace salicylate from protein binding, thereby increasing the free salicylate concentration. We conclude that salicylate in therapeutic concentration can significantly decrease free Danshen concentrations, and Danshen can displace salicylate.

Effect of Chinese medicines Chan Su and Danshen on EMIT 2000 and Randox digoxin immunoassays: wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin measurement by different brands of the same product.

Chan Su is a Chinese medicine prepared from the skin gland of a Chinese toad and is used in treating arrhythmia and other heart diseases. Danshen is prepared from the Chinese medicinal plant and is used for various cardiovascular diseases including angina pectoris. The authors studied the potential interference of such medicines with the widely used EMIT 2000 (Dade Behring; Deerpark, IL) digoxin assay and the recently marketed Randox digoxin assay (Randox Laboratories Ltd, Antrim, United Kingdom) (both run on the Bayer ADVIA 1650 analyzer) (Bayer Diagnostics, Tarrytown, NY) and compared their results with an FPIA (Abbott Laboratories) and a chemiluminescent immunoassay (CLIA; Bayer Diagnostics) for digoxin. Aliquots of drug-free serum were supplemented with 1 microL ethyl acetate extract of Danshen or aqueous extract of Chan Su, and apparent digoxin concentrations were measured by all four digoxin immunoassays (FPIA, EMIT, Randox, CLIA). The authors also supplemented aliquots of several different serum pools prepared from patients taking digoxin with very small amounts of Chan Su or Danshen extract and compared digoxin values with the control digoxin values (serum pool containing no Chinese medicine). The authors observed no interference of Danshen in either EMIT, Randox, or CLIA assay but observed an interference with the FPIA assay. On the other hand, the authors observed high interference of Chan Su in the FPIA assay but moderate interference with the EMIT 2000 and Randox digoxin assays. CLIA assay was again free from any interference. The authors also observed a wide variation in digoxin-like immunoreactivity and magnitude of interference in digoxin immunoassay in different brands of Chan Su and Danshen, indicating poor quality control in manufacturing of these Chinese medicines. Taking advantage of the high protein binding of digoxin-like immunoreactive components of Chan Su, the authors further demonstrated that interference of Chan Su in EMIT 2000 and Randox assays can be mostly eliminated by monitoring free digoxin.

Positive and negative in vitro interference of Chinese medicine dan shen in serum digoxin measurement. Elimination of interference by monitoring free digoxin concentration.

Dan Shen, a traditional Chinese medicine used in the management of cardiovascular diseases, is now available without prescription in the United States from Chinese herbal stores. We demonstrated digoxin-like immunoreactivity of Dan Shen in vitro. Because Dan Shen is used to treat cardiovascular disease, we studied potential interference of Dan Shen with serum digoxin measurement. Addition of microliter quantities of Dan Shen extract to digoxin pools prepared from patients receiving digoxin resulted in falsely elevated serum digoxin concentrations (positive interference) as measured by the fluorescence polarization immunoassay for digoxin (Abbott Laboratories, Abbott Park, IL). More interestingly, serum digoxin concentrations were falsely lowered (negative interference) when measured by the microparticle enzyme immunoassay, also marketed by Abbott Laboratories. Taking advantage of poor protein binding of digoxin (25%) and high protein binding of digoxin-like immunoreactive components of Dan Shen, we further demonstrated that the positive and negative interference of Dan Shen in serum digoxin measurement can be eliminated by monitoring the free digoxin concentration.

[Possibility of using saliva for therapeutic monitoring of digoxin]. / Mozliwosc zastosowania sliny do terapii monitorowanej digoksyny.

Digoxin in salvia and blood serum of 24 patients obtained Bemecor was determined by the method of FPIA (IMx-ABBOT). Mixed saliva was collected by three different types of Salivette (Sarstedt) given in order: normal Salivette with cotton wool swab, Slivette with polyester wool and Salivette with citric acid as a stimulator. It was found, that the correletio between the digoxin concentrations in saliva and serum and saliva/serum rations depended on the type of Salivette. The highest correlation was obtained with the Salivette with polyester wool (r = 0.892), but low concentrations of this drug in serum were good reflected in all samples of saliva, independent on kind of Salivette.

Extraction and quantitation of digoxin and acetyldigoxin from the Digitalis lanata leaf via near-supercritical methanol-modified carbon dioxide.

An extraction process is reported that employs a near-supercritical mixture of CO2 and MeOH to extract the cardiac glycoside, digoxin, from the Digitalis lanata leaf. The method development of the sample preparation procedure is presented in detail, and reasons for trends that occur in the natural products extraction are given.

Determination of cardenolides in hairy root cultures of Digitalis lanata by enzyme-linked immunosorbent assay.

A competitive ELISA technique using digoxin-specific antibody has been developed to determine digoxin and its related compounds in hairy root cultures of Digitalis lanata. The ELISA could detect 0.2-2 nM digoxin and closely related cardenolides. Hairy roots cultured in the dark accumulated very small amounts of cardenolides (0.02-0.07 micrograms/g dry wt), while the content of cardenolides in green hairy roots cultured in the light was increased maximally 600-fold (16.5 micrograms/g dry wt) compared to those in the dark.

Digoxin-like immunoreactivity in Chinese medicine.

Immunoreactive digoxin-like activity was observed in Chinese medicine, KYUSHIN tablet, taken popularly in Japan without prescription. The antibodies used in the assays of digoxin reacted with Chan-su, the major effective component of KYUSHIN, which contained cardiotonic steroids with similar chemical structure as digoxin. One tablet of KYUSHIN had digoxin-like immunoreactivity equivalent to 1.9, 1.5, and 72 micrograms of digoxin measured by three different commercial kits. These different equivalences may be attributed to differences in cross-reactivity of the antibody used in the immunoassays. Two healthy volunteers took two KYUSHIN tablets three times a day, a typical dose, and digoxin-like immunoreactivity reached almost 0.4 micrograms/L in 0.5 day. Therapeutic drug monitoring should be interpreted carefully in patients taking Chinese medicines, many of which contain the Chan-su component.

A digoxin-like immunoreactive substance and atrioventricular block induced by a Chinese medicine "kyushin".

The attempted suicide by 2 women with a kyushin overdose is reported. Kyushin caused them to produce a significant elevation of a serum digoxin-like immunoreactive substance (2.35 and 1.84 ng/ml) and symptoms of nausea, vomiting and general malaise. Their blood biochemistry and electrolytes were normal. In one patient, an electrocardiogram revealed a second degree Wenckebach atrioventricular block and T-wave change. Toad venom, a kyushin ingredient, is possibly responsible for the development of these clinical features and electrocardiographic changes.