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Biomedical subjects

P Datta

Publications and source records attributed to P Datta.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mathematical models to calculate fosphenytoin concentrations in the presence of phenytoin using phenytoin immunoassays and alkaline phosphatase.

Under certain circumstances, it is necessary to measure both fosphenytoin and phenytoin concentrations. We describe equations by which fosphenytoin concentrations can be calculated accurately by using phenytoin immunoassays. We supplemented aliquots of drug-free serum with fosphenytoin and measured the phenytoin equivalent concentrations (reading a) using fluorescence polarization immunoassay and a chemiluminescent assay. Then 10 microL of alkaline phosphatase (ALP) solution was added to the specimen, and after incubation for 5 minutes at room temperature, total phenytoin concentration was measured (reading b). ALP completely converts fosphenytoin to phenytoin in 5 minutes. Therefore, the delta reading (b-a) represents increased observed value due to complete conversion of fosphenytoin to phenytoin for a particular fosphenytoin concentration. By using the x-axis as the delta reading and the y-axis as the target fosphenytoin concentrations, we observed equations that can be used to calculate the concentration of fosphenytoin in the presence of phenytoin. To test the validity of our equations, we prepared 2 serum pools from patients receiving phenytoin and supplemented them with known concentrations of fosphenytoin. Then initial (reading a) and final (after addition of ALP and incubation, reading b) concentrations were measured by immunoassay. We can accurately predict fosphenytoin and phenytoin concentrations from the delta reading.

Alkaline Phosphatase↗
PubMed2000

Evaluation of a centrifuge with rapid turnaround time for the preparation of plasma samples for measurement of common STAT markers on the ACS: 180 system.

Reported is the evaluation of a new centrifugation method, Statspin, that addresses both time and sample separation integrity. The method can successfully separate the plasma fraction from the cellular material in 2 minutes as compared to 20 minutes for the conventional centrifuge method. The Statspin, combined with the ACS:180 system, can generate test results in less than 30 minutes, exclusive of transport to the laboratory. This study demonstrated that the combined technologies offer timing-saving improvements for clinical laboratories offering STAT immunoassays for cardiac markers, endocrine molecules, and therapeutic drugs.

Biomarkers↗
PubMed2000

Folate and vitamin B12 status of adolescent girls in northern Nigeria.

The diets of populations in many developing countries are low in folate and vitamin B12 and a deficiency of either of these vitamins results in increased risk for cardiovascular disease and neural tube defects. The rates of neural tube defects in Nigeria are among the highest reported worldwide. Since many girls marry at an early age in northern Nigeria, we therefore determined the folate and vitamin B12 status of adolescent girls between 12 and 16 years of age in Maiduguri, Nigeria. The mean serum folate concentration for subjects was 15.3 +/- 5.2 nmol/L. Whereas only four subjects (2.4%) had serum folate concentrations lower than 6.8 nmol/L, a level indicative of negative folate balance, 9% of the subjects had serum vitamin B12 concentrations at or below 134 pmol/L, the lower limit of the reference range for their age group. Serum homocysteine was measured in 56 of the 162 subjects and the mean level was 15.9 +/- 5.0 mumol/L. The majority of subjects had serum homocysteine concentrations above the upper limit of the reference range for their age group. We conclude that the adolescent girls we studied were at greater risk for vitamin B12 deficiency than folate deficiency. This conclusion is consistent with the fact that their diet included few foods that contained vitamin B12.

Adolescent↗
PubMed2000

Performance evaluation of a new chemiluminescent cardiac troponin I assay.

Cardiac troponin I is a marker for diagnosis of myocardial damage. Several immunoassays are currently available for determination of concentrations of troponin I in serum. We evaluated a chemiluminescent assay for troponin I using ACS:180 automated analyzer (Bayer Diagnostics). We compared our results with two other immunoassays using the OPUS Magnum (OPUS troponin I assay, Dade Behring) and AxSYM (microparticle enzyme immunoassay, Abbott) analyzers. The within-run and between-run CVs were less than 5% for all three levels of controls. The chemiluminescent assay for troponin I was linear up to a serum troponin I concentration of 50 ng/mL and the detection limit was 0.1 ng/mL of troponin. A good correlation between troponin I concentration measured by the chemiluminescent assay (y axis) and the microparticle enzyme immunoassay (MEIA) (x axis) was observed, although the concentrations of troponin I in individual specimens were approximately four times higher, when measured by the MEIA assay, than those measured by chemiluminescent assay. The correlation coefficient was 0.98 with the regression equation y = 0.22x + 1.125. We also observed a good correlation in troponin I concentrations obtained by the chemiluminescent assay (y axis) and OPUS troponin I assay (x axis). The correlation coefficient was 0.96 and the regression equation was y = 0.79x - 0.52. The correlation coefficient was 0.93 when we compared troponin I concentrations obtained by the OPUS assay (x axis) with the corresponding concentrations obtained by the MEIA assay (y axis). The corresponding regression equation was y = 0.25x + 3.5. We conclude that the chemiluminescent troponin I assay showed good analytical performance.

Humans↗
PubMed1999

Molecular cloning, sequencing, and expression of two late proteins of bacteriophage MB78.

Bacteriophage MB78, a virulent phage of Salmonella typhimurium, does not allow other phages, such as P22 and 9NA, to grow in its presence. A detailed physical map of this phage has been constructed in our laboratory. In an ongoing effort to understand the genetics of this interesting phage, various genes were characterized. Here, we report cloning, sequencing, and expression of two late proteins, coded in a SalI-HindIII fragment (SH9), by using the minicell expression system. Further, we performed a kinetic study of phage proteins by infection the host LT2 cells and compared the proteins produced, with proteins obtained by the minicell expression system. Both sets of proteins run exactly parallel and migrated as 14- and 15-kDa proteins on a polyacrylamide gel. The synthesis of these two proteins started 15 min after infection with MB78 and was prominent after 45 min. One of the proteins exhibited 57% homology to the structural protein of mycobacteriophage L5.

Amino Acid Sequence↗
PubMed1999

Use of alkaline phosphatase to correct the underestimation of fosphenytoin concentration in serum measured by phenytoin immunoassays.

The pharmacologic activity of fosphenytoin, a new phosphate ester pro-drug of phenytoin, is due to in vivo conversion to phenytoin. Fosphenytoin concentrations cannot be accurately estimated by phenytoin immunoassays (fluorescence polarization and chemiluminescence) owing to the nonlinear relation between fosphenytoin concentration and the observed cross-reactivity. The problem of slow conversion of fosphenytoin to phenytoin in serum in vitro can be circumvented by rapidly converting fosphenytoin to phenytoin in vitro by alkaline phosphatase. Drug-free serum, heparin, EDTA, or citrated plasma were supplemented with 2 concentrations of fosphenytoin. Then to 1-mL aliquots of specimen, no enzyme (control), 10 microL, or 25 microL of enzyme solution was added. The specimens were incubated, and phenytoin concentrations were measured by fluorescence polarization and chemiluminescent assays. In the absence of enzyme, we observed little conversion of fosphenytoin to phenytoin, but in the presence of only 10 microL of enzyme, the conversion of fosphenytoin to phenytoin was complete in 5 minutes. We also observed complete conversion of fosphenytoin to phenytoin by alkaline phosphatase in heparin, EDTA, and citrated plasma. If clinically indicated, the phenytoin concentration can be measured before and after addition of enzyme to roughly estimate the rate of conversion.

Alkaline Phosphatase↗
PubMed1999

False-positive troponin I in the MEIA due to the presence of rheumatoid factors in serum. Elimination of this interference by using a polyclonal antisera against rheumatoid factors.

We report false-positive cardiac troponin (cTn) I results in the microparticle enzyme immunoassay (MEIA) using the AxSYM analyzer. We studied serum samples from 12 patients with positive rheumatoid factor but with no indication of myocardial infarction (MI); 2 also had positive antinuclear antibody (ANA) titers. Serum samples from 7 patients with positive ANA titers and negative rheumatoid factors also were studied. Total creatine kinase (CK) was run using a Hitachi 747 analyzer, cTnT using an Elecsys 2010 analyzer, and cTnI and CK-MB using an AxSYM analyzer. We observed no measurable cTnI and cTnT concentrations in 12 control samples or in specimens with positive ANA titers and negative rheumatoid factors. In contrast, samples from 7 of 12 patients containing rheumatoid factors had measurable cTnI concentrations. Four specimens showed cTnI concentrations more than 2.0 micrograms/L, the suggested diagnostic cutoff for MI. None of the specimens showed detectable cTnT. The concentrations of total CK and CK-MB were within normal ranges in all specimens. False-positive results were observed only with the MEIA for cTnI. This interference can be eliminated by using a polyclonal antisera against rheumatoid factor. The chemiluminescent assay for cTnI showed no detectable cTnI concentration in any specimen.

Antibodies, Antinuclear↗
PubMed1999

Porcine endotoxemic shock is associated with increased expired nitric oxide.

OBJECTIVES: Nitric oxide (NO) is believed to decrease systemic vascular resistance in sepsis, but the data are mainly from studies on rats and mice. We tested this hypothesis in pigs and also whether there is induction of the inducible form of nitric oxide synthase (iNOS). DESIGN: Animal study. SETTING: University center. SUBJECTS: Ten pigs. INTERVENTIONS: The pigs were anesthetized and mechanically ventilated. MEASUREMENTS AND MAIN RESULTS: Pulmonary and systemic hemodynamics were monitored and mixed expired NO was measured by chemiluminescence. Animals received 20 microg/kg of endotoxin over 2 hrs. We then infused 25 mg/kg of N(omega)-nitro-L-arginine methyl ester (L-NAME) over 10 mins, followed by 0.5 g/kg of L-arginine, the precursor of NO, for 30 mins more to reverse the effects of L-NAME. Five additional pigs were treated with 20 microg/kg of endotoxin for 2 hrs and followed for another hour. Plasma nitrite/nitrate was measured by Greiss reaction. The animals were then killed and tissues were sampled for iNOS by Western blot, and iNOS messenger RNA by reverse transcriptase polymerase chain reaction. After endotoxin infusion, arterial pressure (BP) initially increased, then decreased to 62+/-1 mm Hg from the baseline of 115+/-4 mm Hg (p<.001). Cardiac output initially decreased, then increased slightly from the baseline of 3.7+/-0.2 to 4.2 +/-0.3 L/min (p<.05). The BP pattern was mirrored by an increase in expired NO concentration from 6.4+/-0.8 to 10.4+/-1.4 parts per billion (p<.05) and increased rate of pulmonary NO excretion in expired gas (VeNO) from 71+/-10 to 146+/-24 pmol/kg/min (p<.05). Inhibition of NOS with L-NAME decreased expired NO concentration and VeNO and increased BP; however, cardiac output decreased. The vasoconstriction produced by L-NAME was partially reversed by L-arginine, and this also increased VeNO from 80+/-18 after L-NAME to 132+/-31 pmol/kg/min (p<.05). Plasma nitrite (n = 5) did not change and there was no iNOS by Western blot analysis in multiple tissues. However, there was a small increase in messenger RNA by reverse transcriptase polymerase chain reaction. CONCLUSIONS: The time course and pattern of changes in expired NO during endotoxemia followed the change in systemic hemodynamics, which supports a causal role for NO in sepsis. However, this is not due to a large production of NO by iNOS induction. The hemodynamic pattern, nitrite in blood, and changes in expired NO also differed markedly from those findings in rodent models and caution should be used in extrapolating from rodents to higher order animals.

Analysis of Variance↗
PubMed1999

Effect of digoxin fab antibody on the measurement of total and free digitoxin by fluorescence polarization and a new chemiluminescent immunoassay.

Digoxin fab antibody (Digibind; Burroughs Wellcome, Research Triangle Park, NC, USA) is used in the treatment of digoxin overdose. The effect of digibind on the measurement of total and free digoxin has been extensively studied. However, the effect of digibind on digitoxin measurements has not been studied thoroughly. The authors studied the effect of digibind on the measurement of total and free digitoxin in vitro using the fluorescence polarization immunoassay and a new chemiluminescent immunoassay. We also studied the capability of digibind to bind digitoxigenin, the major aglycon metabolite of digitoxin. Digibind neutralized both digitoxin and digitoxigenin in vitro, as evidenced by significant reductions in free digitoxin and digitoxigenin (measured as digitoxin equivalent) concentrations. Digibind caused negative interference in the measurement of total digitoxin concentrations by both fluorescence polarization and chemiluminescent assays. However, the magnitude of negative interference was significantly higher with the chemiluminescent assay. For example, in a serum pool supplemented with 80 ng/mL of digitoxin, the concentrations of total and free digitoxin measured by the fluorescence polarization immunoassay were 82.1 ng/mL and 3.3 ng/mL respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 73.9 ng/mL and none detected, respectively. In another serum pool supplemented with 70 ng/mL of digitoxin, the concentrations of total and free digitoxin as measured by the chemiluminescent assay were 69.1 ng/mL and 3.8 ng/mL, respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 29.0 ng/mL and none detected, respectively. Because this effect may also occur in vivo, the progress of Digibind therapy in treating a patient with digitoxin overdose may be monitored by measuring the free digitoxin concentrations.

Cardiotonic Agents↗
PubMed1999

Sensitive methods for determination of free digitoxin concentration using digitoxin immunoassays: demonstration of elevated free digitoxin concentration caused by digitoxin-phenytoin interaction by applying these new techniques.

Digitoxin is very strongly bound to serum albumin. Although free digitoxin is pharmacologically active, it is not monitored because of the lack of a sufficiently sensitive technique. The concentration of free digitoxin in the protein-free ultrafiltrate is usually below the detection limit of digitoxin immunoassays. A modified technique is described by which free digitoxin can be routinely monitored using commercially available immunoassays. The fluorescence polarization immunoassay for determining total digitoxin concentration requires that 100 microL of serum be treated with 300 microL of methanol to precipitate proteins. It is demonstrated that free digitoxin can easily be measured by adding 100 microL of methanol to 300 microL of ultrafiltrate, thus improving the sensitivity of the assay three-fold. The free digitoxin concentration can easily be calculated by dividing the observed value by 3. An attempt to use only ultrafiltrate (no methanol added) caused significant bias in the result, probably as a result of a matrix problem. The chemiluminescent assay for digitoxin does not require any specimen pretreatment and requires only 10 microL of serum. The program was modified and used 50 microL of ultrafiltrate to improve the sensitivity of the free digitoxin assay. If the chemiluminescent assay is used to measure free digitoxin, the true free digitoxin concentration can be calculated by dividing the observed value by 4.3. The free digitoxin concentrations were comparable in eight patients receiving digitoxin as measured by both methods. To show an application of this technique, two serum pools were prepared from patients receiving digitoxin and supplemented with various concentrations of phenytoin. A significant increase in free digitoxin concentration was observed because of the displacement of digitoxin from protein binding sites by phenytoin.

Binding, Competitive↗
PubMed1999

Hemodynamic response to norepinephrine with and without inhibition of nitric oxide synthase in porcine endotoxemia.

The objective of this study was to determine the circuit and cardiac effects of norepinephrine (NE) with and without endotoxin, and how these responses are modified by the inhibition of nitric oxide synthase (NOS). We anesthetized eight pigs and instrumented them for measurements of cardiac output (Q), arterial pressure (Part), and mean pulmonary arterial pressure (Ppa). We also placed a 40-ml balloon in the right atrium for transient obstruction of flow and measurement of the mean circulatory filling pressure (MCFP) and resistance to venous return (RVR). After baseline measurements, animals were treated with 10 microg/kg/h of Escherichia coli endotoxin. At 105 min the measurements were repeated. We then infused 12.5 mg/kg of N(G)-nitro-L-arginine methyl ester (L-NAME) for 10 min and repeated the measurements. At baseline, at the end of endotoxin infusion, and after L-NAME infusion we infused 3, 9, and 27 microg/min of NE for 10 min each, and recorded hemodynamic measurements at each dose. NE shifted the venous return curve (i.e., increased MCFP) to the right without changing RVR, and increased cardiac output (CO) both at baseline and after endotoxin. Endotoxemia markedly flattened the dose-response curves for the change in Part, Ppa, CO, and heart rate with NE. The peak response of Part to NE after endotoxemia was restored with L-NAME, but the other dose-response curves were not affected. NE also did not shift the venous return curve after L-NAME. Furthermore, the increase in Part with NE was of shorter duration after L-NAME than in the baseline condition. In conclusion, NE shifts the venous return curve to the right and improves CO in endotoxic and nonendotoxic conditions. Endotoxemia decreases the arterial responsiveness to NE. L-NAME partly restored this loss of responsiveness in arteries but not in the venous circulation.

Adrenergic alpha-Agonists↗
PubMed1998

Rapid detection of cardioactive bufalin toxicity using fluorescence polarization immunoassay for digitoxin.

Intoxication caused by digitalis-like substances after ingestion of cooked toad soup has been reported. Bufalin, a cardioactive compound, is found in toad. Bufalin is also found in many Chinese medicines. Earlier reports demonstrated cross reactivity of bufalin with fluorescence polarization immunoassay for digoxin. In this report, the authors demonstrated a significantly higher cross reactivity of bufalin with the fluorescence polarization assay for digitoxin. They supplemented aliquots of normal plasma that had various concentrations of bufalin (1 to 50 micrograms/ml) from a local blood bank and measured apparent digitoxin concentrations using fluorescence polarization immunoassay and chemiluminescent assays (ACS digitoxin) for digitoxin. They measured apparent digoxin and digitoxin concentrations using fluorescence polarization, microparticle enzyme immunoassay, and chemiluminescent assays for digitoxin. They observed apparent digitoxin or digoxin concentrations in sera supplemented with bufalin only with the fluorescence polarization assays. For example, the apparent digitoxin concentration observed in a serum supplemented with 25 ng/ml of bufalin was 24.3 ng/ml of digitoxin equivalent. The apparent digoxin concentration observed in the same specimen was 1.33 ng/ml digoxin equivalent. Bufalin caused positive interference in serum digoxin or digitoxin measurements in specimens containing digoxin or digitoxin when concentrations were measured by fluorescence polarization assays. In contrast, bufalin lowered the measured digoxin concentrations in serum pools containing digoxin when digoxin concentrations were measured by the microparticle enzyme immunoassay. The authors conclude that bufalin toxicity can be rapidly detected by the fluorescence polarization assay for digitoxin.

Animals↗
PubMed1998

Bidirectional (positive/negative) interference in a digoxin immunoassay: importance of antibody specificity.

The importance of high specificity in immunoassays used in therapeutic monitoring is highlighted by a case study in which therapeutic-to-toxic borderline digoxin levels were measured by a digoxin immunoassay in the serum sample from a patient administered digitoxin rather than digoxin. The sample, mistakenly sent to the laboratory for digoxin analysis, gave discordant results in three digoxin immunoassays: 1.99 and 0.79 ng/ml in assays using polyclonal antibodies (fluorescence-polarization immunoassay and microparticle enzyme immunoassay, respectively), and <0.1 ng/ml in a chemiluminescent immunoassay using more specific monoclonal antibody. The presence of digitoxin (approximately 40 ng/ml) in the sample was confirmed by three different digitoxin immunoassays. Based on these results, the interference of different levels of digitoxin was studied in the presence of 0, 0.85, 1.9, and 4.7 ng/ml digoxin in all three digoxin assays. The chemiluminescent assay showed no significant interference. The fluorescence-polarization immunoassay showed positive interference in all cases; however, the microparticle enzyme immunoassay showed a bidirectional interference: a positive interference observed at digoxin level <1.8 ng/ml, changing to a negative interference at higher digoxin concentrations. The authors conclude that in countries such as Germany, where both digoxin and digitoxin may be prescribed, caution should be used to interpret digoxin immunoassay results. Digoxin assays, with cross-reactivity to digitoxin <0.1% should be used.

Antibody Specificity↗
PubMed1998

Interference from digitoxin-like immunoreactive factors reduced in a new monoclonal chemiluminescent digitoxin assay.

Endogenous digoxin-like immunoreactive factors (DLIF) can interfere with some digoxin immunoassays. We looked for similar interference, called digitoxin-like immunoreactive factors (DTLIF) in two digitoxin immunoassays: A new chemiluminescent assay (CLIA), processed on the automated random access immunoassay system ACS:180, and a fluorescent polarization assay (FPIA), processed on the semiautomated TDx batch analyzer. One hundred thirty-seven samples of sera were tested from nondigitalized pregnant women, patients with liver or kidney diseases, and cord blood. The CLIA digitoxin assay uses a murine monoclonal antibody and requires no sample pretreatment; the FPIA digitoxin assay uses a polyclonal rabbit antibody and requires sample precipitation. Both assays have a similar dynamic range and sensitivity and give comparable results with commercial controls and external quality control survey samples. Although the CLIA detected no digitoxin in any sample tested, the FPIA showed apparent digitoxin concentrations of more than 2.0 ng/ml for 100% and 44% among cord blood and liver disease specimens, respectively. The highest DTLIF concentration was found in serum from a patient with liver disease (18.1 ng/ml). When spiked with 32 ng/ml digitoxin, six of the samples containing DTLIF generated FPIA digitoxin values of 6% to 27.5% more than the expected digitoxin levels. Two specimens with no detectable DTLIF activity were run as controls, and when spiked with digitoxin, showed target digitoxin concentrations in the FPIA. The CLIA recovered near the target digitoxin values (32 ng/ml) in all spiked samples. It was concluded that the polyclonal FPIA digitoxin assay may give discordant digitoxin concentrations in some patient groups because of interference from digitoxin-like immunoreactive factors. The CLIA digitoxin assay is not affected by DTLIF interference.

Antibodies, Monoclonal↗