A new simplified and rapid method for detection and quantitation of "carbohydrate-deficient transferrin" in serum is described. The method is based on isocratic anion exchange chromatography of isotransferrins in disposable microcolumns followed by a double antibody transferrin radioimmune assay. This technique, which separates all transferrin components isoelectric above pH 5.65, showed a very good reproducibility and accuracy with a coefficient of variation between 5 and 9%. 77 alcoholic patients could be clearly separated from 80 healthy "normal consumers" and 33 total abstainers with a specificity of 100% and a sensitivity of 91%. The values were significantly correlated to the amount of alcohol consumed during the latest month, and declined in abstaining alcoholics with a mean biological half-life of 17 days. Elevated levels occasionally appeared in healthy individuals after daily consumption of 60 g of ethanol during a 10-day period. In a sample of 187 patients with nonalcohol-related conditions only 2% false-positive values were found. This method is suggested as a potential tool for detecting and monitoring alcohol abuse.
To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774‐A.1 macrophage‐like cells. The selection, which was based on intracellular growth competition, rapidly yielded isolates that out‐competed the wild‐type strain during intracellular growth. J774‐A.1 cells responded to challenge with S. typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production. Inhibition of NO production with the use of the competitive inhibitor N‐monomethyl‐ l‐arginine (NMMA) resulted in a 20‐fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth. In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774‐A.1 cells, despite an induction of iNOS mRNA and iNOS protein. Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion‐competent bacteria. The results indicate that S. typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.
We report here a simple method involving urine creatine measurements for testing authenticity and reducing false-negative results in urine testing for drugs of abuse. Urinary creatinine in consecutive patient samples (n = 176) ranged between 0.1 and 31.9 mmol/L (mean 9.8 +/- SD 6.2) and the osmolality in these urines ranged between 49 and 1183 mOsm/kg (mean 595 +/- SD 276). With other consecutive samples in which creatinine was (arbitrarily chosen) less than 4.3 mmol/L (n = 85), the correlation with osmolality was lower. In 10 randomly selected urine samples from different patients, all "clean" for all drugs of abuse in initial immunological drug testing with approved methodology (in which creatinine was less than 4.3 mmol/L and osmolality was less than 200 mOsm/kg), five patients turned out to be drug positive after a simple concentration by volume. In a formerly heavy smoker of cannabis, the excretion of cannabinoids and creatinine was monitored for 93 days. The substances showed very good correlation throughout this period (r = 0.93, P less than 0.001), whereas simple measurements of cannabinoid concentrations would have falsely indicated several relapses of cannabis abuse. Urine samples used in drug-abuse testing should be tested for creatinine; if creatinine is less than 4.0 mmol/L, negative results for drugs may not be valid.
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