Propagation of viral-size particles in lymph and blood after subcutaneous inoculation.

OBJECTIVE: We investigated the propagation of viral-size particles by lymph and blood after subcutaneous injection. METHODS: In the canine model, transport of [99mTc] sulfur colloid particles of different sizes was studied in different settings in venous blood and lymph for 45 minutes after inoculation. RESULTS: The mean arrival time of particles in the blood was 2.10+/-0.46 minutes and 8.87+/-1.72 minutes in the lymph. Lymph flow in the canine leg was 28.79 +/-2.09) microl/min and was increased by leg massage. The particle concentration was 1000 times higher in the lymph fluid than in blood. Particle flux values were comparable in blood and lymph. The accumulation of particles in blood initially rose faster than in lymph. Accumulation in lymph rises slower but continues longer and reaches higher values. Ninety percent of the inoculum remains at the injection site for at least 45 minutes. Particle size matters more in blood distribution. Leg massage enhances particle transport by lymph. CONCLUSIONS: After subcutaneous injection, viral-size particles initially arrive in the blood and later in the lymph. Accumulation in lymph and blood increases for a prolonged time after inoculation. Results suggest possibilities for limiting the spread of infectious matter by early local antiviral treatment.

Electrochemical dehydrogenase-based homogeneous assays in whole blood.

An electrochemical method has been developed for determining NADH in whole blood for dehydrogenase-based assays by flow-injection analysis. NADH generated by dehydrogenase is oxidized by an electron-transfer coupling reagent, 2,6-dichloroindophenol (DCIP). The reduced form of DCIP (DCIPH2) is measured amperometrically by flow-injection analysis. Endogenous interferents were inhibited by p-hydroxymercuribenzoate. Electrode fouling by proteins was not observed under assay conditions. The Emit theophylline enzyme immunoassay and the hexokinase glucose assay were used as models. For the glucose assay, the intraassay CVs were 15% at 0.31 g/L and 3.5% at 1.82 g/L. Recoveries of glucose from whole blood (compared with that for aqueous standards) were 109%, 97.9%, and 101% at 0.050, 2.00, and 5.00 g/L glucose, respectively, and 104%, 101%, and 102% for theophylline at concentrations of 5.0 (low), 16.4 (medium), and 30.2 (high) mg/L, respectively, with corresponding precisions of 12%, 9.5%, and 8.8%. Both assays correlated well with results by reference methods. These studies demonstrate that this method can measure NADH in whole blood without prior separation and that it is potentially applicable to other dehydrogenase-based assays in whole blood.

Platelet activation releases megakaryocyte-synthesized apolipoprotein J, a highly abundant protein in atheromatous lesions.

Apolipoprotein J (apoJ) is an abundant glycoprotein in many biological fluids, and its constitutive high level synthesis is characteristic of many epithelial cells exposed to harsh fluids such as urine, bile, and gastric secretions. In addition, dramatic induction of apoJ occurs in cells surrounding several kinds of pathological lesions. Because platelets and circulating inflammatory cells represent critical elements in numerous pathological processes, we evaluated bone marrow cells for the presence of apoJ. Based upon messenger RNA in situ hybridization and immunofluorescent protein detection, high-level apoJ gene expression and protein accumulation occurred exclusively in mature megakaryocytes. Our results indicate that apoJ is stored in platelet granules and is released into extracellular fluid following platelet activation. Because atheromatous plaque development involves platelet aggregation and activation, we looked for and found abundant apoJ protein in advanced human atheromatous lesions. Thus, platelet sequestration and activation may lead to the rapid deployment of apoJ into sites of vascular injury. We hypothesize that platelet-derived apoJ participates in both short-term wound repair processes and chronic pathogenic processes at vascular interfaces.

Electrochemical homogeneous enzyme immunoassay of theophylline in hemolyzed, icteric, and lipemic samples.

We demonstrate here an electrochemical homogeneous enzyme immunoassay for theophylline, which can be performed in hemolyzed, lipemic, and icteric samples. The assay used an unmodified Syva EMIT theophylline kit. One of the enzymatic reaction products, NADH, reacted with 2,6-dichloroindophenol (DCIP) to reduce DCIP to DCIPH2, which was detected electrochemically with flow-injection analysis. The inter- and intraassay coefficients of variation of this manual technique were < 9% at theophylline concentrations of 14 to 34 mg/L. The CVs were 9-15% at low concentrations (6.3 mg/L), which is below the therapeutic range. Analytical recoveries were 91-97% for normal serum and 92-111% for hemolyzed, icteric, or lipemic sera. The measured concentrations (y) were compared with those obtained by the fluorescence polarization immunoassay (x); a scatter plot of the results showed a linear relationship of y = 1.00 x - 0.57 mg/L (r = 0.966, Sy/x = 1.51). This alternative way to measure the serum concentration of theophylline overcomes the shortcomings of spectrophotometric methods, by which it is difficult to measure theophylline in severely hemolyzed, icteric, or lipemic sera.

Structure and stability of apolipoprotein J-containing high-density lipoproteins.

Apolipoprotein J (apoJ) defines a heterogeneous subclass of human plasma high-density lipoproteins (HDL) having a bimodal distribution of molecular mass of 70-90 kDa (approximately 50%) and 200 kDa or larger (approximately 50%). ApoJ-HDL are unstable in stored plasma, and must be evaluated within 24 h. All apoJ-HDL in freshly obtained plasma have alpha 2 electrophoretic mobility and are distinct from a minor subpopulation of apoAI-HDL which electrophorese in the pre beta region. Although apoAI is not associated with the majority of plasma apoJ-HDL, a small fraction of these particles also containing apoAI. There is little variation in the apoJ/apoAI mole ratio of apoJ-HDL immunoaffinity purified from the same individual on different days. In addition, there is a constant ratio among individuals, assessed for five volunteers, of 4.9 +/- 0.6. Purified apoJ added directly to apoJ-depleted plasma can interact with apoAI or with apoAI-containing lipoproteins, as evidenced by the association of apoAI with apoJ that is reisolated by immunoaffinity chromatography. The amount of apoAI associated with apoJ increases linearly with increasing amount of apoJ added, over the range of apoJ concentrations tested. No other known apolipoprotein is associated with apoJ. By two-dimensional electrophoretic analysis, the lipoproteins containing both apoJ and apoAI have approximate molecular masses of 350-400 kDa. Taken together, the results suggest that the interaction between apoJ and apoAI is physiologically important and that lipoproteins which contain both apoJ and apoAI can be produced in the plasma. ApoJ-HDL and apoJ/apoAI-HDL may have different functions and metabolic fates or may represent different stages of apoJ catabolism.

Effects of nisoldipine on the no-reflow phenomenon in globally ischemic rat hearts.

Isolated working rat hearts, receiving no drug treatment, recovered low values of contractile function after 33 min of zero-flow global ischemia and 30-min reperfusion, had lower ATP and creatine phosphate (CrP) values in the subendocardium than in the subepicardium after reperfusion (subendocardial/subepicardial ratio ATP = 0.44, CrP = 0.45), and had a subendocardial reperfusion defect including 15.9% of ventricular cross-sectional area. Contracture pressure increased during the later part of ischemia to 25.4 mm Hg. Addition of nisoldipine (1 nM) 10 min before ischemia did not depress preischemic contractile function and did not delay or reduce contracture or the breakdown of high-energy phosphate compounds during ischemia. On reperfusion, nisoldipine-treated hearts showed a dramatic improvement in recovery of contractile function, increased subendocardial energy levels yielding a more uniform transmural energy distribution (subendocardial/subepicardial ratio ATP = 0.79, CrP = 0.94), and enhanced reflow to the subendocardium (area of no reflow = 1.0%). In contrast, addition of vehicle or a low concentration of verapamil (1 nM) before ischemia or nisoldipine during reperfusion did not improve recovery of contractile function. The beneficial effects do not appear to be induced by direct myocardial actions of nisoldipine but may reflect improved vascular function which is associated with the vascular selectivity of this calcium antagonist.

Sequestration electrochemistry: the interaction of chlorpromazine and human orosomucoid.

A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K0ass was determined as 0.39 X 10(6) M-1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.

Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level.

Electrochemical enzyme immunoassay methodology has been developed to take advantage of the selectivity of antibody reactions, the amplification feature of an enzyme-based assay, and the ease with which small amounts of the enzyme-generated product can be detected electrochemically. A heterogeneous sandwich enzyme immunoassay was used in this work as the model assay. In this type of assay, the antigen is sandwiched between the enzyme conjugate and a primary antibody that is adsorbed to the solid phase. Alkaline phosphatase is a suitable enzyme for electrochemical assays since it catalyzes the conversion of electroinactive phenyl phosphate to electroactive phenol. The product, phenol, is then quantitated by liquid chromatography with electrochemical detection in a thin-layer flow cell with a carbon paste electrode at 0.895 V vs Ag/AgCl. The current produced by the oxidation of phenol is directly proportional to the analyte (antigen) concentration. The problem associated with these types of solid-phase immunoassays is that the adsorption of the primary antibody is desired while the adsorption of other assay proteins is not. The detection limits are generally defined by the ability to control this nonspecific adsorption. The detection limit of a previous electrochemical assay for rabbit IgG was 100 pg/ml and was limited by a large background current observed in the absence of antigen. In the present study, each step of the assay was examined in order to determine the sources of this background current, and it was found that the major contribution was from the nonspecific adsorption of the enzyme conjugate. Using combinations of Tween 20 and bovine serum albumin as blocking agents, the level of nonspecific adsorption was reduced by 96%.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome studies in human subjects chronically exposed to arsenic in drinking water.

A two-year study was carried out on human subjects of various ages and backgrounds who had been drinking water containing more than 0.05 mg/liter (0.05 ppm) arsenic for a period of at least five years. The main aim was to correlate the frequency of chromosome aberrations and sister chromatid exchanges in the lymphocytes with the amount of arsenic in the water. In addition, we explored the incidence of skin cancer, fetal wastage, and genetic or developmental abnormalities. Several other variables--eg, coffee, wine, and cigarette consumption; sex; residence (rural vs urban); and exposure to chemicals, smelters, or pesticides--were also taken into consideration. The data on chromosome aberrations (104 exposed and 86 control individuals) and on sister chromatid exchanges (98 exposed and 83 control individuals) did not show that arsenic at concentrations used by our population (greater than 0.05 mg/liter) has any effect on these parameters. Similarly, no other health effects of arsenic at these concentrations were found.

Gene frequencies in the domestic cats of Reno, Nevada: confirmation of a recent hypothesis.

On the basis of a sample of 156 animals, allele frequencies for the cat population of Reno, Nevada have been estimated. Comparison of this population with populations of 22 other cities shows that Reno's cats are more similar to those of northeastern North America than to those in the southwest. This is consistent with a recent hypothesis that suggests there were two distinct European sources of North American cats, and that present gene pools in North American cities depend primarily on their history of early settlement.