A reflectance photometer with a square photodiode array detector for use on multilayer dry-film slides.

This semiautomated prototype reflectance photometer measures reflected light from multilayer dry-film slides. The instrument makes use of a square photodiode array detector, a Hewlett-Packard desktop computer, and a modified mechanical transport mechanism from an Ektachem DT60 analyzer. When 2 microL of serum is placed on a dry-film slide, a colored spot is formed. The slide is automatically transported to an incubation area and then to the photometer area. There the spot is illuminated with dual tungsten lamps, and the reflected light passes through an interference filter, where it is focused on a square photodiode array containing 10,000 individual detectors. The analog signal from each detector is digitized and transmitted to a computer for calculation of the percentage of reflectance. I used a series of algorithms to locate the spot, estimate spot area, correct for minor variations in sample volume, and compute the average reflectance from a central spot area. To evaluate the instrument's performance, I ran parallel glucose determinations in the Beckman Astra; results correlated well. The small sample size along with no dead sample volume makes the system useful for small sample volumes.

Automated enzymatic method for determining ammonia in plasma, with 14-day reagent stability.

This is an automated, cost-efficient enzymatic method for determining ammonia in plasma. In most assays for ammonia the reagents are stable for only one or two days at 4 degrees C, but the reagents in our method are stable for at least 14 days. Our method was developed for use with a Cobas-Fara analyzer but can be easily adapted to other automated analyzers. It is essential to use closed sample vials. Our results, obtained at much less cost, correlate well with those by the Du Pont aca method. Precision studies gave the following results: within-run (n = 20 each), for x = 46 mumol/L, SD = 0.7 mumol/L, CV = 1.6%, and for x = 593 mumol/L, SD = 4.0 mumol/L, and CV = 0.7%; day-to-day precision during a year: for x = 47.7 mumol/L, SD = 2.4 mumol/L, CV = 5.0% (n = 360); and for x = 643 mumol/L, SD = 20.5 mumol/L, and CV = 3.2% (n = 365). The standard curve was linear to 1000 mumol of NH3 per liter.

Time course of response of individual messenger RNAs in the rat heart to T3.

The time course of response of specific mRNAs following administration of triiodothyronine (T3) to hypothyroid rats was examined. We were particularly interested in identifying mRNAs showing a rapid response. Hypothyroid rats were injected with 0.2 mg of T3/100 g body wt and total cardiac RNA was prepared 0.5, 1, 2, 3, 5, 12 and 24 h later. RNA was translated in vitro in the presence of [35S]-methionine, the labeled peptides separated by two-dimensional electrophoresis and quantitated by digital matrix photometry. Of a total of 427 translational products 13 were identified to be selectively responsive to thyroid hormone. A specific mRNA coding for a protein designated as spot 72b (Mr 81,600, pI 5.34) was observed to show the most rapid response to T3. Administration of T3 to the hypothyroid animal resulted in an increase in the level of spot 72b by 2.6-fold within 1 h. The lag time between injection of T3 and response of other specific mRNA species varied between 5 to 24 h. These results demonstrate the diversity of response of individual cardiac mRNAs. The specific T3 responsive mRNA species described in the heart have not been demonstrated in other tissues indicating that induction of distinctive mRNA species is highly tissue specific. Relatively late responses may represent indirect effects of T3 mediated by interaction with other hormonal or metabolic signals. The rapid induction of spot 72b suggests it may result from the interaction of T3 with the nuclear receptor leading to a direct effect on the expression of this gene in the heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Ischemia of the dog heart induces the appearance of a cardiac mRNA coding for a protein with migration characteristics similar to heat-shock/stress protein 71.

Recent evidence indicates that different forms of stress, including hypoxia, can induce specific proteins called heat-shock or stress proteins in various types of mammalian cells. These studies examined whether myocardial ischemia can result in increased levels of proteins with molecular weight and isoelectric point characteristics similar to those described for heat-shock or stress proteins. The left anterior descending coronary artery of the dog heart was completely occluded; normal and ischemic myocardial samples were obtained 6 hours after occlusion; and total cardiac proteins and RNA were prepared. Ribonucleic acid was translated in vitro in a modified rabbit reticulocyte lysate system, and [35S]-methionine-labelled translational products as well as unlabelled cardiac proteins were separated by two-dimensional gel electrophoresis. Total proteins were visualized by silver staining and in vitro translation products quantified by fluorometry. A translatable mRNA coding for a 71,000 dalton peptide with an isoelectric point of 5.8 was markedly increased in the ischemic myocardium after 6 hours of ischemia. A protein with similar migration characteristics was detected in ischemic myocardium but not in normal myocardium. These results indicate that an mRNA coding for a translational product with similar migration characteristics of heat-shock protein 71 is induced by ischemia in the dog heart.

Rapid effects of insulin on in vitro translational activity of specific mRNA in diabetic rat heart.

We studied the time course of response of specific cardiac mRNA after administration of insulin to diabetic rats. The primary aim was to identify specific cardiac mRNA, which show a rapid response to insulin administration. Diabetic rats were injected with 2 U of regular insulin intravenously, and total cardiac RNA was prepared 0.5, 1.5, 3, 5, 12, and 24 h later. RNA was translated in vitro in the presence of [35S]methionine and the translational products separated by two-dimensional electrophoresis and quantitated by digital matrix photometry. A rapid change in the translational activity of five specific mRNA species was observed within 0.5 h after administration of insulin to the diabetic animal. One translational product exhibits a more delayed response at 1.5 h. The predominance of three of these products was increased, while that of three was decreased. Two specific mRNA coding for translation products designated as spots 97 and 106 show the most significant change, with a dramatic decrease of 15-fold and 6.5-fold, respectively, within 0.5 h after insulin administration. The change in levels of these specific mRNA species could result from effects of insulin at various sites of mRNA synthesis or degradation. However, the rapidity of the response is compatible with a direct effect of insulin on gene expression. The very quick response of these specific mRNA species to insulin could thus serve as a useful model system to examine the molecular mechanisms of insulin action in the heart.

Diabetes-induced alterations in the translational activity of specific messenger ribonucleic acids isolated from rat hearts.

During diabetes mellitus, total proteins and ribonucleic acids are significantly decreased in the rat heart, and these parameters can be increased by insulin administration. To determine whether all ribonucleic acids are equally sensitive to insulin, we examined the influence of this hormone on individual translatable ribonucleic acids. Cardiac ribonucleic acid prepared from control, untreated, and insulin-treated diabetic animals was translated in vitro in the presence of [35S]methionine. The radiolabeled peptides were separated by two-dimensional gel electrophoresis and were analyzed by fluorometry. We found that diabetes induces both qualitative and quantitative changes in the predominance of a few specific translatable messenger ribonucleic acid species. The translation of 11 messenger ribonucleic acid species was significantly decreased and that of eight messenger ribonucleic acid species was significantly increased in diabetic preparation. Twelve of the 19 translation products were quantified by digital matrix photometry: three labeled peptides were observed only when cardiac ribonucleic acid from diabetic animals was added to the cell-free translation system, four new peptides appeared when cardiac ribonucleic acid from control animals was added, and although the remaining five peptides were translated in vitro after either control or diabetic ribonucleic acid was added, their relative predominance was altered 2- to 200-fold. When translation products coded for by messenger ribonucleic acids prepared from either diabetic or hypothyroid hearts were compared, we found that most of the alterations induced by diabetes were also induced by hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

A cost-effective system for performing therapeutic drug assays. I. Optimization of the theophylline assay.

We have developed a cost-effective system for performing drug assays by using the COBAS BIO centrifugal analyzer and EMIT reagents. With the theophylline assay as a model, we were able to optimize both instrument and chemical parameters to allow assay of 2470 samples with a 100-test EMIT kit. This decreased current reagent costs from +2.00 per test for the Syva manual method to 9 cents per test for our proposed method. Within-run and between-run CVs are typically less than 3% and 6%, respectively.

Determination of serum aspartate aminotransferase with pyridoxal 5'-phosphate in the Technicon SMAC and Du Pont aca compared and correlated with the IFCC recommended method.

Inexpensive improvements in continuous-flow analytical apparatus can eliminate some perplexing inconsistencies and limitations in the use of the Technicon SMAC instrument for measuring the activity in serum of aspartate aminotransferase (EC 2.6.1.1) in the presence of pyridoxal 5'-phosphate. In addition, a calibration procedure based on values for this enzyme obtained by a modified IFCC method can be used to calibrate both the Technicon SMAC and the Du Pont aca instruments to produce excellent correlation between the two.

High-resolution densitometry: analysis of stained albumin bands as a model for electrophoresis of serum proteins.

In discussing the principles of quantitative analysis in thin-layer media, we show that requirements for quantitative analysis are not satisfied when stained protein electrophoretic bands are scanned with a conventional rectangular-slit densitometer. We investigated a high-resolution densitometer based on a linear photodiode array as an alternative analytical tool, using stained electrophoretic bands of radio-labeled human serum albumin as a simplified model for results of serum protein electrophoresis. Identical protein bands scanned with both the high-resolution densitometer and a conventional densitometer were quantified with improved accuracy and precision by the new instrument. We also used the high-resolution densitometer to develop a computer model for performance characteristics of a rectangular-slit densitometer.

Design of a microcomputer-based densitometer system: application to serum protein electrophoresis.

We describe how a microcomputer (Apple II Plus) can be interfaced with a commercial densitometer to provide control functions, do automated data processing, and print final reports. The amplified densitometer photodetector signal is digitized, stored, and plotted on the cathode-ray tube to provide a real-time graphical display of data acquisition. The software was designed to identify automatically the five main serum protein fractions and to compute final results. Algorithms were designed to provide medically relevant comments for most of the common abnormal protein patterns, and the comments section was designed so that it could be modified to use specific comments composed by the user. A final report, printed in duplicate, contains information such as the patient's name, identifying numbers, numerical results, normal values, medically relevant comments, and a digitized high-resolution plot of the electrophoretic pattern. The densitometer was one that already had been in use for several years; the microcomputer system, software, and interfaces cost less than $2200.

Multilayer film analysis for glucose in 1-microL samples of plasma.

With the ultramicroanalytical system described here we can measure glucose in 1 microL of plasma or serum. The sample is placed on a dry, multilayer film element (Eastman Kodak), where a colored spot about 3.5 mm in diameter develops. The reflectance of these spots is measured with a reflectance digital matrix photometer that was conceived, designed, and constructed in our laboratory. The spot is illuminated with monochromatic light and its image is projected by a camera lens onto the photosensitive surface of a linear photodiode array containing 512 individual photodetectors. The photodetector signals are processed by a computer to obtain the reflectance and diameter of the spot. The latter is proportional to sample volume. Because the reflectance of the spot does not depend greatly on sample volume, accurate pipetting is not required. The coefficients of variation of repeatable glucose analyses were, for 400, 3000, and 5120 mg/L, 1.7, 2.3, and 2.8%, respectively. The correlation coefficient (r) between glucose analyses by our method (y) and with the Ektachem 400 (x) was 0.9918; the regression equation was y = 1.07x - 94.3 mg/L.

Polyclonal 3,5,3'-triiodothyronine (T3) antibodies in a euthyroid woman and their effect on radioimmunoassays for T3.

Thyroid function tests in a 17-yr-old euthyroid woman with a slightly enlarged thyroid gland were normal, except for an apparently elevated serum T3 level which was over 8 times greater than the upper limit of normal, when her serum was directly analyzed for T3 by an antibody-coated tube RIA method. When T3 analysis was performed with a RIA procedure that also used serum directly and separated free and bound radiolabeled hormone by polyethylene glycol precipitation, an absurd and conflictingly low result was obtained in which the bound fraction from the patient's serum contained 22% more radiolabeled T3 than the bound fraction from the zero T3 standard. These results suggested the presence of endogenous T3 antibodies which interfered with these RIA procedures. Further studies revealed the presence of IgG-kappa and IgG-lambda polyclonal antibodies that bound T3, but not T4, which accounted for the artifactual elevation in T3. The T3 antibodies were also detected by agarose electrophoresis of serum labeled with tracer quantities of [125I]T3. Analysis for T3 by RIA on Sephadex or RIA of an ethanol extract of the patient's serum both eliminated interference from these endogenous T3 antibodies. The T3 autoantibodies occurred in this patient even though there was no previous history of thyroid medication. Thus, endogenous thyroid hormone-binding antibodies produce artifactually abnormal results with RIA methods that assay serum directly, whereas inactivation and removal of the immunoglobulins by alcohol extraction or Sephadex obviates interference from such proteins.

Reflectance digital matrix photometry.

We have designed and constructed a digital matrix photometer for quantitative measurement of reflected light of small chromophoric areas or colored spots. The areas are divided conceptually into small subunits in which the reflected light is measured. This is done through stepwise scanning with a photodiode array. The sum of the individual reflectance measurements is related to the total amount of an analyte in the spot. We have found that this technique makes possible accurate and precise quantitation of light-absorbing substances in small areas of different sizes, shapes, and internal irregularity measuring as little as 1 mm across.

Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart.

The influence of thyroid hormone on the translational activity of specific cardiac mRNA was determined by in vitro translation of RNA isolated from the heart of normal, hypothyroid, and 3,3',5-triiodo-L-thyronine-injected hypothyroid rats. Proteins synthesized in vitro in the presence of [35S]methionine were separated by two-dimensional gel electrophoresis and quantitated by a novel scanning procedure using digital matrix photometry. A total of 421 translational products were detected by fluorography and changes in the predominance of 12 of these were influenced by the thyroid state of the animals. The relative predominance of 8 species was increased in euthyroid animals, whereas 4 translational products were increased in hypothyroid animals. The majority of these thyroid hormone-related alterations occurred in spot pairs of similar molecular weights, but slightly different isoelectric points. In contrast, the relative predominance of mRNAs coding for the major contractile proteins, light chain 1, light chain 2, tropomyosin, actin, and myosin heavy chain was not altered by the thyroid status of the animals. The relative levels of these abundant mRNA species remained unaltered in spite of a thyroid hormone-related increase in total RNA levels. In vivo effects of thyroid hormone on cardiac RNA levels are complex. In addition to a general increase in total RNA and mRNA levels, increases or attenuations in the predominance of a small number of specific mRNA species are observed when euthyroid and hypothyroid animals are compared.

Fetal hemoglobin: optimum conditions for its estimation by alkali denaturation.

Alkali denaturation is the most commonly used technic to estimate fetal hemoglobin in red blood cells. The Betke and colleagues method (Nature 184: 1959; 1877-78) using cyanmethemoglobin (HiCN) was recommended by an International Committee for Standardization in Hematology for fetal hemoglobin levels between 2 and 40%. We showed that precision with samples containing up to 5% fetal hemoglobin can be considerably improved by measuring the absorbance of HiCN at 420 nm rather than at 540 nm because the molar absorptivity is 10 times greater in the Soret band. To determine optimum conditions for the assay, we studied the kinetics and stoichiometry of conversion of hemoglobin to HiCN with various concentrations and mixtures of ferricyanide, kinetics of alkali denaturation, ammonium sulfate precipitation of denatured HiCN, filtration as opposed to centrifugation for separating denatured HiCN, stability of HiCn in water and KCN solutions, and linearity of absorbance in the Soret band. With our modified procedure, the CV with normal amounts (less than 1%) of fetal hemoglobin improved from 28% to 8.7%. The CVs with 6.8% and 55% fetal hemoglobin were 7.0-4.4% respectively, and linear estimates were obtained with cells containing up to 50% fetal hemoglobin. We conclude that our modified alkali denaturation procedure yields reliable and reproducible estimates of fetal hemoglobin over a wide range of concentrations.