Comparing different statistical methods for evaluating diagnostic effectiveness of clinical tests: respiratory distress syndrome as a model.

Phospholipids in specimens of amniotic fluid from 346 patients were quantified and the results evaluated in light of the clinical outcome. Fifty-eight neonates had respiratory distress syndrome. We used this data base to compare different statistical methods for evaluating test effectiveness and diagnostic discrimination. Dichotomizing quantitative tests into binary tests with arbitrary cutoff values was inadequate for comparing test effectiveness. Subgrouping the data into deciles and calculating the incidence of respiratory distress syndrome for each decile avoided the problems of the preceding approach and was easy to calculate and comprehend; however, this method lacked statistical power. Relative operating characteristic curves yielded more statistical power, but results were more difficult to calculate and were not intuitively obvious to most workers in the laboratory. A modified cumulative frequency plot, combining elements of both decile subgrouping and relative operating characteristic curves, was easily calculated and intuitively obvious. These plots, like relative operating characteristic curves, provided an index for quantifying test effectiveness. When used in combination with standard cumulative frequency curves, they also provided direct diagnostic information on disease probability for any value of the clinical assay.

Evaluating the clinical effectiveness of amniotic fluid assays in predicting respiratory distress syndrome in the neonate.

Amniotic fluid phospholipids from 346 patients' specimens were quantified and evaluated against the clinical outcome (i.e., respiratory distress syndrome or normal respiratory function). Concentrations of lecithin, sphingomyelin, phosphatidylglycerol, and the lecithin/sphingomyelin reflectance ratio were evaluated by ordered frequency distribution and stepwise discriminant function analysis. The lecithin/sphingomyelin ratio was the best single test for discriminating between respiratory distress syndrome and normal pulmonary function in the fetus, slightly superior to lecithin assay alone. A combination of lecithin/sphingomyelin ratio and lecithin concentration, however, appeared to optimize the discriminant function, although the clinical significance of this test combination remained marginal. High concentrations of phosphatidylglycerol were correlated with high concentrations of lecithin, and virtually ruled out respiratory distress syndrome. Absence of phosphatidylglycerol was not diagnostic. High concentrations of sphingomyelin increased the probability of respiratory distress syndrome. We suggest the following stepwise series of tests to optimize diagnosis: phosphatidylglycerol concentration, sphingomyelin concentration, and finally lecithin-sphingomyelin ratio.

Evidence for a special relationship between proteolysis and single cell necrosis.

A high rate of single cell necrosis is a common phenomenon in neoplastic and preneoplastic lesions, accounting for growth rates that are significantly less than the cell birth rate. We present data relating the process of protein turnover to single cell necrosis. Cells were labeled with 3H-leucine and 14C-thymidine; the loss of radioactivity from the cell protein and DNA was then measured for 3-6 days. Preliminary experiments showed that cell necrosis by freeze-thawing cells did not significantly contribute to the degradation of cell proteins. Similar results were observed with dying 3T3-SV40 cells at high density. L-cells, however, showed a progressive increase in cell loss as higher cell densities were attained on the monolayer. Although proteolysis remained constant in the culture, analysis of the cells recovered from the high density monolayers showed little loss of labeled protein after adjustment for loss of label in the DNA. Three possible explanations are proposed: DNA turns over with cell protein (unlikely), single cell necrosis involves a special mechanism that facilitates reutilization of amino acids, or single cell necrosis includes only cells that are selectively involved in protein turnover. A unique relationship between single cell necrosis and proteolysis is suggested.

Protein synthesis and degradation in growth regulation in rat embryo fibroblasts: role of fast-turnover and slow-turnover protein.

Cultured rat embryo fibroblasts, when stimulated to grow by the addition of fresh medium containing 10% serum, showed an increase in synthesis of slow-turnover proteins while maintaining a uniform degradation rate for these proteins. Slow-turnover proteins with a half-life of 2.4 days accounted for approximately 95% of the cell protein, while the remaining protein could be described in terms of two fast-turnover pools. When we labeled cells to limiting levels over a period of 4 days, the fast-turnover pools became undetectable; with 2-hour labeling periods, however, 25% of the label entered the fast-turnover pools. Fibroblasts, stimulated to grow by fresh growth medium, showed proportionate and coordinate increases in synthesis of both fast-turnover and slow-turnover proteins during the growth period, both returning to baseline levels on reaching the new steady state. No changes could be detected in degradation of either pool during growth. Fibroblasts placed in a serum-free medium showed a decrease in cellular protein and an increased degradation of slow-turnover proteins, while degradation of fast-turnover proteins remained unchanged. We conclude that the slow-turnover protein pool forms the bulk of the cell proteins and turns over at a fairly constant rate. Growth stimulation is effected almost entirely by stimulation of protein synthesis in this pool, while decreasing cellular protein growth is a result of enhanced degradation within this pool.

Evidence of heterogeneity of protein-turnover states in cultured cells.

Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847--859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, 'chased' for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267--283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.

Role of lysosomes in protein turnover: catch-up proteolysis after release from NH4Cl inhibition.

Cultured rat embryo fibroblasts, when placed in media with 10% serum containing 20 mM NH4Cl, show an inhibition of protein degradation and, concurrently, an accumulation of numerous, large vacuoles, partially filled with cellular debris. Cells placed in a serum-free media exhibit an enhanced degradation of cell protein, which is also inhibited by NH4Cl. When these cells are removed from media containing NH4Cl and placed in fresh media, the material accumulated in these vacuoles is rapidly and quantitatively released to the media in both an acid-soluble and acid-insoluble from. NH4Cl inhibits rapidly and specifically the lysosomal proteolytic mechanism, and is without effect on the basal turnover mechanism. The lysosomal proteolytic mechanism accounts for approximately 25% of protein turnover, and, at least in low density cultures, can be stimulated to levels which account for more than half of the protein turnover in the cell. The major pathway for the degradation of fast turnover proteins appears to be separate from lysosomal mechanism.