David Porter

BACKGROUND: Ovarian failure is a common toxic effect of chemotherapy. Studies of the use of gonadotropin-releasing hormone (GnRH) agonists to protect ovarian function have shown mixed results and lack data on pregnancy outcomes. METHODS: We randomly assigned 257 premenopausal women with operable hormone-receptor-negative breast cancer to receive standard chemotherapy with the GnRH agonist goserelin (goserelin group) or standard chemotherapy without goserelin (chemotherapy-alone group). The primary study end point was the rate of ovarian failure at 2 years, with ovarian failure defined as the absence of menses in the preceding 6 months and levels of follicle-stimulating hormone (FSH) in the postmenopausal range. Rates were compared with the use of conditional logistic regression. Secondary end points included pregnancy outcomes and disease-free and overall survival. RESULTS: At baseline, 218 patients were eligible and could be evaluated. Among 135 with complete primary end-point data, the ovarian failure rate was 8% in the goserelin group and 22% in the chemotherapy-alone group (odds ratio, 0.30; 95% confidence interval [CI], 0.09 to 0.97; two-sided P=0.04). Owing to missing primary end-point data, sensitivity analyses were performed, and the results were consistent with the main findings. Missing data did not differ according to treatment group or according to the stratification factors of age and planned chemotherapy regimen. Among the 218 patients who could be evaluated, pregnancy occurred in more women in the goserelin group than in the chemotherapy-alone group (21% vs. 11%, P=0.03); women in the goserelin group also had improved disease-free survival (P=0.04) and overall survival (P=0.05). CONCLUSIONS: Although missing data weaken interpretation of the findings, administration of goserelin with chemotherapy appeared to protect against ovarian failure, reducing the risk of early menopause and improving prospects for fertility. (Funded by the National Cancer Institute and others; POEMS/S0230 ClinicalTrials.gov number, NCT00068601.).

CONTEXT: High dietary phosphorus intake has deleterious consequences for renal patients and is possibly harmful for the general public as well. To prevent hyperphosphatemia, patients with end-stage renal disease limit their intake of foods that are naturally high in phosphorus. However, phosphorus-containing additives are increasingly being added to processed and fast foods. The effect of such additives on serum phosphorus levels is unclear. OBJECTIVE: To determine the effect of limiting the intake of phosphorus-containing food additives on serum phosphorus levels among patients with end-stage renal disease. DESIGN, SETTING, AND PARTICIPANTS: Cluster randomized controlled trial at 14 long-term hemodialysis facilities in northeast Ohio. Two hundred seventy-nine patients with elevated baseline serum phosphorus levels (>5.5 mg/dL) were recruited between May and October 2007. Two shifts at each of 12 large facilities and 1 shift at each of 2 small facilities were randomly assigned to an intervention or control group. INTERVENTION: Intervention participants (n=145) received education on avoiding foods with phosphorus additives when purchasing groceries or visiting fast food restaurants. Control participants (n=134) continued to receive usual care. MAIN OUTCOME MEASURE: Change in serum phosphorus level after 3 months. RESULTS: At baseline, there was no significant difference in serum phosphorus levels between the 2 groups. After 3 months, the decline in serum phosphorus levels was 0.6 mg/dL larger among intervention vs control participants (95% confidence interval, -1.0 to -0.1 mg/dL). Intervention participants also had statistically significant increases in reading ingredient lists (P<.001) and nutrition facts labels (P = .04) but no significant increase in food knowledge scores (P = .13). CONCLUSION: Educating end-stage renal disease patients to avoid phosphorus-containing food additives resulted in modest improvements in hyperphosphatemia. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00583570.

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1'F) and P453L (p1/p6 PP5'L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity approximately 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.

Abstract We have found that d-amino acid oxidase is rapidly and irreversibly inhibited by cyanide during oxidative turnover of the carbanion of nitroethane. The inhibited enzyme has an absorption spectrum which is characteristic of a covalent flavin-substrate adduct. The following summarizes the results and interpretation of experiments which establish the mechanism of cyanide inhibition as well as the chemical mechanism of oxidation of nitroalkanes by d-amino acid oxidase. 1. The kinetic mechanism of oxidation of the nitroethane carbanion (S-) to form acetaldehyde (P), hydrogen peroxide, and nitrite, was deduced from a combination of stopped flow and O2-monitored kinetic measurements and is the following. [see PDF for equation] Evaluation of all the rate and equilibrium constants showed that the major pathway for flavin oxidation is the oxidation of ErP by O2. 2. The rate of inhibition of the enzyme by cyanide is regulated by κ2, and O2 is not consumed during the inhibition process. Since the rate of cyanide inhibition increases as the O2 concentration is raised, cyanide does not react with ErP or with a species in rapid equilibrium with ErP. Consequently cyanide must react rapidly with an intermediate, EX, which is formed from E0S in a reaction, or reactions, controlled by κ2. This is depicted kinetically as follows: [see PDF for equation] The inhibited enzyme, EI, does not react with substrate or O2 under the conditions, and within the time scale, of routine kinetic experiments. 3. The inhibited enzyme (EI) contains, per FAD, 1 eq each of substrate and cyanide, but is lacking the nitro group. 4. Treatment of EI with hot methanol produces a free flavin-substrate adduct in good yield and with no irreversible spectral or chemical modification. At 70° and pH values greater than 8 the free flavin-substrate adduct releases cyanide and is readily converted (l10 min) under aerobic conditions to FAD, acetaldehyde, and, presumably, H2O2. Anaerobically, FADH2 is produced. These solvent-catalyzed reactions of the adduct in large part mimic the enzyme-catalyzed oxidation of the substrate. 5. The spectral and ionization properties, as well as the chemical reactivity, of the free flavin-substrate adduct closely resemble those of 5-substituted dihydroflavins in general, and those of 5-cyanomethyl-1,5-dihydroflavin in particular. For these reasons we assign the structure 5-cyanoethyl-1,5-dihydro-FAD to the free flavin-substrate adduct. The kinetic turnover mechanism, the locus of action of cyanide, and the structure of the free flavin-substrate adduct, taken together, enable us to propose a detailed chemical mechanism for the oxidation of nitroethane carbanion by d-amino acid oxidase. E0S is a noncovalent complex in which the substrate carbanion is sufficiently close to the flavin (presumably the N5 position) to perturb the electronic properties of the latter. Attack of the carbanion at N5 of the flavin is controlled by κ2 and results in the formation of 5-nitroethyl-1, 5-dihydro-FAD. Elimination of nitrite forms a highly reactive cationic imine (EX) at the N5 flavin position to which solvent adds to [see PDF for equation] form a carbinolamine. Finally, FADH2, is eliminated from the carbinolamine, leaving acetaldehyde (P) noncovalently bound to Er. Cyanide attacks the cationic imine (EX) in competition with solvent to form 5-cyanoethyl-1,5-dihydro-FAD (EI). This adduct is not reactive with O2 under conditions normally used to study the enzyme.

Horseradish peroxidase catalyzes the anaerobic oxidation of 2-nitropropane (probably as the nitronate R-) by H2O2 via Compounds I and II to form R-R. The oxidation rate is stimulated 10-fold by O2 and the products become acetone and NO-2. The aerobic oxidation of R- is a free radical chain reaction which is initiated by peroxidase and propagated by R because (a) R-R, reasonably, must arise as 2R leads to R-R, (b) over 90% of the enzyme-initiated reaction occurs free in solution, and (c) the Km value for R- is independent of the type of initiator. We present a scheme for initiation, propagation, and termination which explains product structure, the effects of resorcinol, CN-, ascorbate, superoxide dismutase, and catalase, as well as the ping-pong reaction kinetics. We used methanenitronate to investigate the locus of electron transfer from R- into the heme moiety of the enzyme because this donor reacts with Compound II to form an isoporphyrin, which then rearranges to a modified enzyme in which the ferriheme contains the nitromethyl group in covalent linkage at a methine carbon. The modified enzyme is 30-50% as active catalytically as the native enzyme. We argue that reduction of Compound II by R- occurs at the methine carbon by two competing pathways, namely, direct one electron transfer and, at about one-half the frequency, homolytic cleavage of a covalent heme-substrate adduct (isoporphyrin).