Electronic absorption, EPR, and resonance raman spectroscopy of CooA, a CO-sensing transcription activator from R. rubrum, reveals a five-coordinate NO-heme.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

Identification of two important heme site residues (cysteine 75 and histidine 77) in CooA, the CO-sensing transcription factor of Rhodospirillum rubrum.

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Stability of ceftazidime and amino acids in parenteral nutrient solutions.

The stability of ceftazidime was studied under conditions simulating administration via a Y-injection site into a primary infusion of parenteral nutrient (PN) solution; the stabilities of ceftazidime and amino acids when the drug was added directly to PN solutions were also studied. Three PN solutions containing 25% dextrose were used; the amino acid contents were 0, 2.5%, and 5%. Ceftazidime with sodium carbonate was used to prepare stock solutions of ceftazidime 40 mg/mL in both 0.9% sodium chloride injection and 5% dextrose injection; to simulate Y-site injection, samples were added to the three PN solutions to achieve ceftazidime concentrations of 10 and 20 mg/mL, or 1:1 and 1:3 ratios of drug solution to PN solution. Samples of these admixtures were assayed by high-performance liquid chromatography (HPLC) initially and after room-temperature (22 degrees C) storage for one and two hours. Additional solutions were prepared by adding sterile water for injection to ceftazidime with sodium carbonate; drug solutions were added to each PN solution in polyvinyl chloride bags to achieve ceftazidime concentrations of 1 and 6 mg/mL. The samples were assayed by HPLC for ceftazidime concentration after storage at 22 degrees C for 3, 6, 12, 24, and 36 hours and at 4 degrees C for 1, 3, 7, and 14 days. Amino acid stability was analyzed in admixtures containing 5% amino acids and ceftazidime 6 mg/mL after 24 and 48 hours at 22 degrees C and after 7 and 10 days at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of famotidine 20 and 40 mg/L and amino acids in total parenteral nutrient solutions.

The stability of famotidine in total parenteral nutrient (TPN) solutions and the concentrations of amino acids in the presence of famotidine were determined. Two famotidine concentrations (20 mg/L and 40 mg/L) and two amino acid concentrations (20 g/L and 42.5 g/L) were studied under the following storage conditions: refrigerated for 24 hours and then kept at room temperature (20-22 degrees C) for 24 hours, at room temperature for 48 hours, or refrigerated for seven days. Control TPN solutions were studied under the same storage conditions. TPN solutions also contained dextrose 25%, electrolytes, trace elements, and vitamins. Famotidine concentration was determined at 0, 24, and 48 hours and at seven days by high-performance liquid chromatography. Amino acid concentration was determined in the TPN solutions containing 42.5 g/L of amino acids without famotidine and with famotidine 40 mg/L under both 48-hour storage conditions. At 24 hours, all solutions retained at least 95% of the initial famotidine concentration. Seven of the eight famotidine solutions retained more than 95% of the initial famotidine concentration at 48 hours. All samples refrigerated for seven days retained more than 95% of the initial famotidine concentration. The concentration of amino acids in TPN solutions containing 42.5 g/L of amino acids was not affected by the addition of famotidine 40 mg/L under either 48-hour storage condition. Famotidine in concentrations of 20 mg/L and 40 mg/L is stable under the studied 48-hour storage conditions in TPN solutions containing amino acid concentrations of either 20 g/L or 42.5 g/L.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of ranitidine admixtures frozen and refrigerated in minibags.

The stability of ranitidine hydrochloride stored frozen and refrigerated in polyvinyl chloride minibags was studied. Ranitidine hydrochloride was added to either 5% dextrose injection or 0.9% sodium chloride injection to yield concentrations of 0.5, 1.0, and 2.0 mg/mL. In phase 1 of the study, admixtures containing ranitidine hydrochloride 1 mg/mL were stored at 4 degrees C for 10 days. In phase 2, solutions were frozen for 30 days at -30 degrees C and were later refrigerated for 14 days. Ranitidine concentration was tested using a stability-indicating high-performance liquid chromatographic assay at time zero and at intervals during storage. Sterility tests were performed on some samples, and various admixtures were visually inspected and tested for pH. At least 90% of the initial concentration of ranitidine remained in all solutions at all storage conditions. No visual changes or changes in pH or sterility were observed. Ranitidine hydrochloride in concentrations of 0.5, 1.0, and 2.0 mg/mL in 5% dextrose injection or 0.9% sodium chloride injection may be stored in polyvinyl chloride minibags frozen for 30 days followed by refrigeration for an additional 14 days.

Stability of ranitidine hydrochloride and amino acids in parenteral nutrient solutions.

The stability of ranitidine hydrochloride in parenteral nutrient (PN) solutions and the effect of ranitidine hydrochloride on the amino acids in the PN solutions were studied. Six PN solutions (three each with amino acid contents of 2.125 and 4.25%) were prepared. Each PN solution also contained dextrose 25%, electrolytes, trace elements, vitamins, and heparin sodium. Ranitidine hydrochloride injection was added to four of the PN samples. Of the final samples, two contained no ranitidine, two contained ranitidine hydrochloride 50 micrograms/mL, and two contained ranitidine hydrochloride 100 micrograms/mL. Admixtures of ranitidine hydrochloride at the two concentrations in 0.9% sodium chloride injection were also prepared. Samples were observed for color change and tested for pH during storage at room temperature. Concentrations of amino acids were measured after 24 hours in samples without ranitidine and in samples containing ranitidine hydrochloride 100 micrograms/mL. Ranitidine hydrochloride content was determined by high-performance liquid chromatography at 12, 24, and 48 hours. No visual changes or pH changes occurred by 24 hours. All PN solutions became darker by 48 hours. The presence of ranitidine hydrochloride did not substantially affect amino acid concentrations. At 24 hours, at least 90% of the initial ranitidine concentrations remained in all samples. In three of the four PN samples at 48 hours, less than 90% of initial ranitidine concentrations remained. Ranitidine hydrochloride in concentrations of 50 and 100 micrograms/mL in parenteral nutrient solutions containing 4.25 and 2.125% crystalline amino acids is stable for 24 hours at room temperature. Under these conditions, concentrations of the amino acids contained in the PN solutions were not affected by the addition of ranitidine hydrochloride.

Total parenteral nutrition with F080 in cirrhotics with subclinical encephalopathy.

It has been proposed that hepatic encephalopathy and malnutrition in cirrhosis can be reversed by infusion of a protein formula (F080) enriched with branched-chain amino acids (valine, leucine, isoleucine) and containing decreased amounts of aromatic amino acids (phenylalanine, tyrosine, tryptophan). This hypothesis was tested by measuring changes in encephalopathy status, plasma ammonia, amino acid profile, and liver function during seven metabolic balance studies in three patients with cirrhosis and subclinical encephalopathy given increasing amounts (20-100 g/d) of F080. The results showed the following: 1) positive nitrogen balance was achieved only with 80 and 100 g F080/day; 2) plasma ammonia fell during negative, but increased during positive nitrogen balance; 3) plasma tyrosine and cystine fell significantly (p less than 0.05) with all intakes of F080; 4) the abnormal branched-chain to aromatic amino acid ratio was reversed; 5) extracellular volume was expanded in all patients; 6) albumin, bilirubin, prothrombin time became abnormal; and 7) encephalopathy did not significantly change from baseline. It is concluded that, in this population, F080 is an inadequate nutritional formula when given as the sole protein source because it produces hypotyrosinemia and hypocystinemia. The marked changes in the ratio of branched-chain to aromatic amino acids are not accompanied by improvement in encephalopathy.