A physician's office-based digoxin assay (Seralyzer) evaluated for interference by endogenous digoxin-like immunoreactive factors.

A digoxin test for a physician's office based-chemistry analyzer (Ames Seralyzer) was evaluated for possible interference by digoxin-like immunoreactive factors (DLIF). Sera from patients likely to have high concentrations of DLIF (renal and hepatic patients, pregnant women, and neonates) as well as from normal patients and umbilical cord blood were analysed by the Seralyzer digoxin immunoassay and by a fluorescence polarization digoxin immunoassay (Abbott TDx) known to detect DLIF. For all patients who were not taking digoxin (n = 85) only four patients (4.7 percent) measured apparent digoxin values greater than 0.2 ng per mL by the Seralyzer compared to 64 (75 percent) by the TDx analyzer. Measurements of DLIF from adrenal extracts demonstrated a 17-fold greater potency for detection of DLIF by the TDx (2.9 ng per mL) compared to the Seralyzer technique (0.18 ng per mL). However, recovery data suggest that the presence of digoxin reduces the potency of DLIF interference as a function of increasing digoxin concentrations especially for the TDx assay. This diminished DLIF crossreactivity in the presence of digoxin is one explanation for the comparable correlation observed for both non-renal and renal failure patients taking digoxin when measured by these two immunoassays.

Interference of the chemotherapeutic agent etoposide with the direct phosphotungstic acid method for uric acid.

A 62-year-old woman receiving chemotherapy with etoposide showed discrepant uric acid values as measured by a direct phosphotungstic acid (PTA) method (150 mg/L) compared with a uricase technique (40 mg/L). After ultrafiltration, the positive interference for the direct PTA method was retained in the protein fraction, but not in the filtrate. Adding exogenous etoposide to drug-free serum confirmed this interference for the direct PTA method, but not for the uricase procedure or a PTA technique preceded by dialysis. Decisions for aggressive patient management are often based on the magnitude of hyperuricemia. We do not recommend that the direct phosphotungstic acid method be used to measure uric acid in patients receiving etoposide.

A monoclonal antibody to the carboxyterminal domain of procollagen type I visualizes collagen-synthesizing fibroblasts. Detection of an altered fibroblast phenotype in lungs of patients with pulmonary fibrosis.

Excessive collagen deposition plays a critical role in the development of fibrosis, and early or active fibrosis may be more susceptible to therapeutic intervention than later stages of scarring. However, at present there is no simple method for assessing the collagen-synthesizing and secreting activity of fibroblasts in human tissues. Type I procollagen carboxyterminal domains are proteolytically removed during collagen secretion. Thus, antibodies to these domains should stain fibroblasts synthesizing type I collagen but not extracellular collagen fibrils which could mask the signal from the cells. We developed and characterized a monoclonal antibody (Anti-pC) specific for the carboxyterminal propeptide of type I procollagen. To determine the relationship between Anti-pC staining and collagen synthesis, we stained embryonic and adult chicken tendon. Embryonic chick tendon fibroblasts actively synthesizing type I collagen stained heavily with Anti-pC, while quiescent adult tendon fibroblasts did not stain with Anti-pC. Wounded adult tendons developed fibroblasts that stained with Anti-pC at the wound site. Thus, Anti-pC specifically visualized fibroblasts actively synthesizing collagen. Lung biopsies from patients with fibrotic lung disease were stained with Anti-pC. Interstitial and intraalveolar fibroblasts in biopsies from patients with active fibrosis stained intensely with Anti-pC, while normal human lung was unstained. The absence of staining in normal lung supports the hypothesis that fibrosis is associated with an altered collagen-synthesizing phenotype of tissue fibroblasts. Anti-pC may provide a useful clinical tool for assessing fibrogenic activity at sites of tissue injury.

The effects of monensin on transport of membrane components in the frog retinal photoreceptor. I. Light microscopic autoradiography and biochemical analysis.

We have explored the use of the Na+-H+ ionophore monensin as a potential tool for the investigation of membrane assembly and transport in retinal photoreceptors. Autoradiographic analysis of frog retinas incubated with [3H]leucine in the presence of monensin revealed a lack of concentrated silver grains ("bands") at the base of the rod outer segments, in contrast to controls. This is indicative of a pronounced monensin-induced decrease in disc membrane assembly. Biochemical analyses of whole retinas and isolated rod outer segment membranes showed that protein synthesis (including opsin synthesis) was not significantly inhibited under these conditions, whereas passage of membrane protein to the rod outer segment was blocked. Glycerolipid synthesis was not significantly affected by monensin. The results suggest that membrane proteins (e.g., opsin) destined for incorporation into the rod outer segment must pass through the Golgi apparatus and demonstrate the potential utility of monensin for inhibiting aspects of marcomolecule transport in photoreceptors.

The effects of monensin and of puromycin on transport of membrane components in the frog retinal photoreceptor. II. Electron microscopic autoradiography of proteins and glycerolipids.

Monensin converts the Golgi apparatus of rod photoreceptors into distended vacuoles, similar to those seen in other monensin-treated cell types, and leads to the accumulation of [3H]leucine in the distended vacuoles. As evaluated by quantitative, electron microscopic autoradiography, transport of newly made proteins--both to the outer segments and to the presynaptic terminals--is inhibited. These effects suggest that the Golgi apparatus is involved in transport in both principal directions within the highly polarized photoreceptors, a matter of interest since there seems to be only a single, extensive, Golgi apparatus in the cell body. Seemingly there are two distinguishable "sorting" routes, for proteins, out of the Golgi apparatus and, for the terminals, an additional non-Golgi route. Accumulation of newly made glycerolipids in the outer segments and terminals is less affected by monensin than is accumulation of new proteins, and glycerolipid accumulation is little affected by puromycin, an inhibitor of protein synthesis. These latter findings suggest that the routes or mechanisms of assembly of newly made lipids into membranes in the photoreceptors are at least partially dissociable from those for newly made proteins.

Effects of monensin on photoreceptors of isolated frog retinas.

Monensin induces the vacuolization of the Golgi apparatus in photoreceptors of isolated frog retinas and also, more slowly, produces a vacuolization of the pre-synaptic terminals. Accompanying these effects is an inhibition of transport of protein to the outer segment so that the radioactive bands normally detectable by autoradiography do not form. Monensin thus promises to be a useful tool in the study of intracellular transport in photoreceptors. The findings reported here indicate that impairment of the functioning of the Golgi apparatus considerably diminishes transport of membrane protein to the rod outer segment suggesting that passage through the Golgi apparatus is an obligatory step for completion of outer segment membrane or its transport to the outer segment.