Enzyme immunoassay for the quantitation of human immunoglobulin G specific for the glycolipid of Enterobacteriaceae

An enzyme immunoassay (EIA) is reported that quantitates human immunoglobulin (Ig)G specific for the glycolipid (Re-GL) of Salmonella minnesota Re 595. Re-GL was adsorbed to polystyrene microcuvettes, and 100 microliters of appropriately diluted lots of human plasma or affinity-purified rabbit anti-Re-Gl IgG was added to the cuvettes. The cuvettes were incubated, washed, and then reacted with Staphylococcus aureus protein A-peroxidase. After a final wash the substrate 2,2'-azino-di(3-ethylbenzthiazoline)sulfonic acid (ABTS) was added to yield a blue-green color with a maximum absorption at 405 nm. Using the affinity-purified rabbit anti-Re-IgG as standard, it was possible to quantitate specific levels of anti-Re-GL. The EIA was rapid (2 hr), with an absolute sensitivity of 5 ng of specific anti-Re-GL IgG and an interassay coefficient of variation of 24%. On a unit-mass basis, rabbit anti-Re-GL IgG had 10-fold greater activity than comparable affinity-purified human anti-Re-GL IgG from normal donor plasma. When a rabbit standard was used, the mean level of specific anti-Re-GL IgG in lots of pooled human plasma, which consisted of 18,330 liters, was 1.1 +/- 0.28 micrograms/ml. Of 237 individual donors tested, 24 (10%) had levels of anti-Re-GL IgG that were 10-fold or greater than the mean of 1.1 micrograms/ml. The EIA provided a rapid, reproducible method for accurate quantitation of specific anti-Re-GL IgG in the screening of large volumes of human plasma.     

Mechanism of inhibition of red blood cell glutathione reductase activity by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea).

A study of the mechanism of inhibition of glutathione reductase activity by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) demonstrated that the enzyme was protected from inhibition by mercaptoethanol. N-Ethylmaleimide competes with BCNU and also protects the enzyme from inhibition. The inhibited enzyme showed decreased affinity for NADPH. Our data suggest that the inhibition of glutathione reductase is due to an alteration of the redox state. Several other red cell enzymes were inhibited by high concentrations of BCNU; these enzymes were also protected by mercaptoethanol. Among alkylating agents tested, only BCNU inhibited glutathione reductase at therapeutic levels.     

Glyoxalase enzyme system in human muscular dystrophy.

Glyoxalase I and glyoxalase II activities were determined in skeletal muscle of control subjects and of patients with Duchenne dystrophy, other major forms of muscular dystrophies and certain neuromuscular disorders. The glyoxalase I activity was normal in all diseases examined except in Duchenne and limb girdle types of muscular dystrophy, where it showed a significant moderate decrease. The glyoxalase II activity in normal human muscle was very low, and the activity was unaltered in muscle of patients with Duchenne and other major forms of muscular dystrophies and spinal muscular atrophy. The selective decrease of glyoxalase I activity in recessively inherited muscular dystrophies may have some relevance to some phases of these disease processes.     

A radioenzymatic assay for free and conjugated normetanephrine and octopamine excretion in man

A radioenzymatic method for the measurement of free and conjugated normetanephrine (NMN) and octopamine (OCT) is described for human urine. The assay is based on the conversion of NMN or OCT to radiolabeled metanephrine (MN) or synephrine (SYN) by phenylethanolamine-N-methyl transferase (PNMT), using tritium-labeled S-adenosyl-methionine [³H]SAM as methyl donor. Thin-layer Chromatographie separation yields an assay of high specificity. The sensitivity of the assay is 5 and 2.5 pg of NMN or OCT, respectively.The normal value of free and conjugated NMN was found to be 23 ± 13 and 102 ± 49 ng/mg creatinine. Four patients with pheochromocytoma had highly increased levels of free and conjugated NMN. The urinary excretions of free and total octopamine were 5.7 ± 2.8 and 34.8 ± 16.6 ng/mg of creatinine, respectively, in normal patients.     

Stress-induced analgesia in the mouse: strain comparisons

Inescapable footshock is capable of differentially activating opioid- and non-opioid-mediated mechanisms of stress-induced analgesia in the rat, depending on the temporal and intensive parameters of its administration. In this study we compared the effects of opioid and non-opioid stress analgesia in two strains of mice, one known to be deficient in central opioid binding sites (CXBK) and one normal in this regard (C57BL/6BY). We found that although the C57 strain showed robust opioid and non-opioid stress analgesia, the CXBK strain only showed stress analgesia of the non-opioid type.     

Correlation of E. coli K-1 bacteremia and capsular polysaccharide antigenemia in acute and chronic infection

The K-1 polysaccharide is an important virulence factor in human $\text{E. coli}$ infections. Using $\text{E. coli}$ 016K1, we investigated the kinetic association of bacteremia and K-1 antigenemia in acute lapine and canine infections and in a chronic infection model of neutropenic rats. Additionally, we assessed the presence of K-1 antigenemia in $\text{E. coli}$ K-1 bacteremic patients. K-1 was measured by a solid phase radioimmunoassay (RIA) using cross-reactive equine anti-Group B meningococcal IgM. In acute infections, none of the dogs or rabbits developed antigenemia even with a bacteremia of 2 × 10⁴ CFU/ml or 5 × 10⁵ CFU/ml, respectively. Antigenemia appeared in the rabbit only with an infecting dose of ? 5 × 10⁸ CFU. In the rat model we observed an initial bacteremia of 10³ CFU/ml, which increased to 10⁶ CFU/ml at 24 hrs. However, antigenemia was most often delayed, appearing in only ?30 hrs postinfection. Percent mortality was directly associated with the degree of bacteremia and antigenemia. In acute human $\text{E. coli}$ K-1 bacteremia, 11 of 22 (50%) of patients were positive for K-1 antigenemia. The data demonstrated that K-1 polysaccharide was not usually detectable in the early stages of bacteremia, but occured only after prolonged infection or very high infecting doses. The RIA to measure K-1 antigenemia would not be a useful diagnostic tool.     

Increased catecholamine excretion after labetalol therapy: a spurious effect of drug metabolites

Patients with essential hypertension were treated for four weeks with the alpha- and beta-adreno-receptor blocking agent labetalol. Urinary excretion of total catecholamines, metanephrine plus normetanephrine and vanillylmandelic acid was measured with various methods before and during treatment. An unidentified substance interfering with the fluorimetric method for catecholamines and the photometric assay for metanephrines caused falsely high values of those substances. Using appropriate methodology no changes of total catecholamines, metanephrine plus normetanephrine and vanillylmandelic acid excretion were found after labetalol therapy. Our findings are important in preventing errors in the diagnosis of pheochromocytoma as well as in the evaluation of the effects of labetalol on the sympathetic nervous system in man.     

Isospora infection in a homosexual man

A case of Isospora belli in a homosexual male is described. The procedures used for recovery and staining the cyst stages of Isospora are included.     

3-Mercaptopyruvate sulfurtransferase (EC 2.8.1.2): a simple assay adapted to human blood cells

A product of cysteine catabolism, 3-mercaptopyruvate, is enzymatically degraded to sulfur and pyruvate by a sulfurtransferase (EC 2.8.1.2.) present in human red and white blood cells. A simple sulfurtransferase assay is reported which takes advantage of the fact that pyruvate is conveniently measured enzymatically by lactate dehydrogenase (LDH) after the elimination of 3-mercaptopyruvate, also a substrate of LDH, by addition of N-ethylmaleimide. Sulfite is employed as sulfur acceptor, and conditions for a reproducible assay including data for preparation and storage of reactants, their assay, and their optimal concentrations are given. The apparent K_m for sulfite is 6.5 · 10^?3 M and for 3-mercaptopyruvate is 1.9 · 10^?3 M. A reducing agent, in this assay dithiothreitol (Cleland's reagent), is essential for active transsulfuration. A normal metabolite, 3-mercaptopyruvate is reported elsewhere to have the capacity of producing polyploidy and chromosomal endoreduplication, a feature rendering it of interest in tumor metabolism.     

In vitro activity of newer beta-lactam agents in combination with amikacin against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens

The in vitro activity of cefoperazone, ceftazidime, ceftizoxime, moxalactam, and N-formimidoyl thienamycin was evaluated alone and in combination with amikacin to assess possible synergistic activity against isolates of amikacin-resistant Pseudomonas aeruginosa and multidrug-resistant Serratia marcescens and Klebsiella pneumoniae susceptible to amikacin (one S. marcescens isolate was also resistant to amikacin). The checkerboard agar dilution method was used. Ceftazidime and thienamycin followed by moxalactam and cefoperazone were the most active agents versus the P. aeruginosa alone and in combination testing. Ceftazidime, moxalactam, and thienamycin showed the greatest activity against S. marcescens, and all agents except cefoperazone were active against K. pneumoniae. The finding of synergy or partial synergy in combination testing was found in the majority with all three genera, including levels below the breakpoint for both amikacin and the beta-lactam agents. This wide in vitro activity indicates that clinical evaluation of these agents in treatment of multidrug-resistant infections is warranted.     

Semi-automated competitive protein binding analysis of serum thyroxine on reusable Sephadex columns and its advantages over radioimmunoassay.

Competitive protein-binding analysis of serum thyroxine on small, reusable, Sephadex columns has been further studied and improved. The improved, semi-automated procedure results in reduced working time and costs. It has also been established that triiodothyronine crossreacts only 1/6 to 1/9 as well as thyroxine, and can be ignored because it represents only about 1/80 of the total serum iodothyronine content. The economic and methodological advantages of the improved method over radioammunoassay and other displacement assays are discussed.     

Changes in serum high density lipoproteins in women on oral contraceptive drugs

Serum lipids, lipoproteins, and lipoprotein subfractions were measured in a group of 18 women aged 20 through 39 who were users of oral contraceptive drugs, and in 19 age-matched controls. Concentrations of the major lipid and lipoprotein classes were higher in the users, but the elevation was statistically significant only in the case of the high density lipoproteins. This increase was shown to be due to a highly significant increase (275 ± 9 vs. 223 ± 9 mg/100 ml, p < 0.005) in the denser high density lipoprotein subfraction (HDL₃). Levels of the other subfraction (HDL₂) were similar in users and controls. Thus, anovulatory steroids have selective effects on individual types of high density lipoproteins. Studies of such specific effects may help to further define the functional properties of the high density lipoproteins such as their apparent protective role in atherosclerosis.     

Radioimmunoassay for human pancreatic secretory trypsin inhibitor: measurement of serum pancreatic secretory trypsin inhibitor in normal subjects and subjects with pancreatic diseases

A reliable radioimmunoassay (RIA) for human pancreatic secretory trypsin inhibitor (PSTI) has been developed. The method is highly sensitive (0.4 ng/ml), reproducible and specific. A good parallel relationship was observed between the standard curve and dilution curves for serum and urine. The PSTI bound to trypsin-alpha 2-macroglobulin complexes was found not to be immunoreactive, whereas a part of the psti-trypsin complex was immuno-reactive. In healthy individuals, serum PSTI level ranged from 5.4 ng/ml to 16.0 ng/ml, the average being 11.3 ng/ml (S.D. +/- 2.7). Elevated values were observed in patients with acute pancreatitis (highest value 3200 ng/ml), and in some patients with chronic relapsing pancreatitis.     

Polyamine distribution in cellular compartments of blood and in aging erythrocytes.

Human blood was separated into pure preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets, and platelet free plasma. The mean concentrations of putrescine, spermidine, and spermine per 10(9) cells were found to be several orders of magnitude higher for leukocytes than erythrocytes. There was no significant difference between leukocyte types. Platelets and plasma had relatively low levels in proportion to the amounts contributed by erythrocytes and leukocytes to whole blood. Human erythrocytes were age-separated by density and the changes in polyamine concentrations in maturing erythrocytes were documented. There were highly significant statistical differences between young and old red blood cells for putrescine, spermidine and spermine. The clinical use of red blood cell polyamines as an indicator of the activity of the bone marrow in anemic states is suggested.     

Biphasic reaction kinetics in an anomalous isozyme of erythrocyte pyruvate kinase.

A mutant erythrocyte isozyme of pyruvate kinase (PK) (ATP: pyruvate phosphotransferase, EC 2.7.1.40) has been found in associatin with chronic hemolytic anemia in two siblings who were doubly heterozygous for the isozyme and for quantitative PK deficiency of the usual form. The isozyme was characterized by approximately normal maximum reaction velocities but had markedly decreased affinity for the substrate, phosphoenolpyruvate (PEP), with 5-fold to 10-fold increases in half-saturation constants and decreased interaction between substrate binding sites. Two distinctly separable kinetic patterns for PEP were observed with multiple specimens. Concentrations of fructose 1,6-diphosphate (FDP) required for half-maximal activation were two orders of magnitude greater than controls, and optimal pH was lowered to 6.5. stability at 4 degrees C was markedly decreased, but the lost enzyme could be reactivated by fdp for periods even longer than normal controls.     

Naloxone does not affect pain relief induced by electrical stimulation in man

We wished to determine if pain relief that resulted from transcutaneous (TNS) or spinal cord electrical stimulation in patients with chronic pain was due to activation of an endogenous opiate-related pain control system. Naloxone (0.4–10 mg) or saline was injected in double-blind fashion intravenously into opiate-naive subjects with chronic pain who achieved 30% or greater pain relief with spinal cord stimulation (4 patients) or TNS (9 patients). Subjects rated their pain during stimulation and 2, 5, 10 and 15 min after the injection. Two days or more later the procedure was repeated using the alternate agent (naloxone or saline). Naloxone did not decrease the pain relief induced by stimulation, and therefore the effects of stimulation are probably not mediated by the endogenous opiates.     

Tryptophan loading may reverse tolerance to opiate analgesics in humans: A preliminary report

Five patients on chronic opiate medication to treat low-back and leg pain were determined to have developed opiate tolerance on the basis of their failure to obtain significant relief (rated on a subjective pain scale and by the degree of straight leg-raising they were able to endure) after receiving 30 mg of morphine administered i.v. in divided doses over 35 min. After these patients' diets had been supplemented with 4 g/day of l-tryptophan for 2–9 weeks, they achieved significant relief from pain when the opiate tolerance test was re-administered, and were able to lead more active lives while reducing their daily opiate intake. Chronic opiate administration probably reduces the serotonin turnover rate in the central nervous system; it may be that this is reversed by loading with the serotonin precursor, l-tryptophan.