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PubMed2001

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine.

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.

Animals↗
PubMedDate not supplied

Gas-chromatographic method for the halothane metabolites, trifluoroacetic acid and bromide, in biological fluids.

A simple and rapid method is presented for the quantitative determination of bromide and trifluoroacetic acid in urine, plasma or serum. The biological fluid is treated with dimethyl sulfate in an acidic medium, resulting in the formation of the methyl ester of trifluoroacetic acid and methyl bromide. The volatile derivatives are then isolated from the samples via a head-space technique, separated and resolved by gas chromatography, and detected by flame ionization. Detection of the two metabolites is linear within the sample concentrations studied. The method is reproducible and applicable to the determination of the two metabolites in biological fluids in the 0.1-10 mM concentration range. The method is comparable to previous techniques but is less time consuming, and both bromide and trifluoroacetic acid can be determined simultaneously. Preliminary pharmacological studies of urine and blood metabolite levels in rats and human patients anesthetized with halothane agree with existing results determined by other methods. In addition, the results indicate that bromide concentrations in human blood remain elevated for a much longer period of time than those in rat blood.

Anesthesia↗
PubMed1975

Amino-acid sequence of lac repressor from Escherichia coli. Isolation, sequence analysis and sequence assembly of tryptic peptides and cyanogen-bromide fragments.

The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion-exchange chromatography, gel filtration and preparative thin-layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl-Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19. The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N-terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication.

Amino Acid Sequence↗
PubMed1984

Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides.

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.

Amino Acid Sequence↗
PubMed1972

Temperature dependence of chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells.

1. The temperature dependence of the steady-state self-exchange of chloride between human red cells and a plasma-like electrolyte medium has been studied by measuring the rate of (36)Cl(-) efflux from radioactively labelled cells. Between 0 and 10 degrees C the rate increased by a factor of eight corresponding to an Arrhenius activation energy of 33 kcal/mole.2. The rate of chloride exchange decreased significantly in experiments where 95% of the chloride ions in cells and medium were replaced by other monovalent anions of a lyotropic series. The rate of chloride self-exchange was increasingly reduced by bromide, bicarbonate, nitrate, iodide, thiocyanate, and salicylate. The latter aromatic anion was by far the most potent inhibitor, reducing the rate of chloride self-exchange to 0.2% of the value found in a chloride medium.3. The temperature sensitivity of the chloride self-exchange was not affected significantly by the anionic inhibitors. The Arrhenius activation energies of chloride exchange were between 30 and 40 kcal/mole in the presence of the six inhibitory anions mentioned above.4. The rate of self-exchange of bromide, thiocyanate, and iodide between human red cells and media was determined after washing and labelling cells in media containing 120 mM bromide, thiocyanate, or iodide respectively. The rate of self-exchange of the three anions were 12, 3, and 0.4% of the rate of chloride self-exchange found in the chloride medium.5. The Arrhenius activation energies of the self-exchange of bromide, iodide, and thiocyanate were all between 29 and 37 kcal/mole, the same magnitude as found for the self-exchange of chloride.6. Although approximately 40% of the intracellular iodide and salicylate ions appeared to be adsorbed to intracellular proteins, the rate of tracer anion efflux followed first order kinetics until at least 98% of the intracellular anions had been exchanged.7. The self-exchange of salicylate across the human red cell membrane occurred by a different mechanism than the one utilized by the inorganic monovalent anions. The activation energy of salicylate exchange (13.2 kcal/mole) was significantly lower than that of inorganic anion exchange. Salicylate exchange increased with decreasing pH in contrast to the exchange of chloride, which decreases when pH is lowered.

Adsorption↗
PubMed1977

Methyl bromide as a microbicidal fumigant for tree nuts.

Methyl bromide (MeBr) has broad microbicidal activity, but its use as a disinfectant for food is limited by the resulting bromide residues. Increasing the MeBr concentration, exposure temperature, or exposure period of a treatment tended to increase both the microbicidal efficacy of MeBr and the bromide residues. Its sporicidal activity was less at high than at low relative humidity within the range of 20 to 99%. Both the efficacy and the resulting residues of a MeBr treatment varied inversely with the load of product in a fumigation chamber due to sorption of the fumigant. Fumigation tests with almond kernels inoculated with Escherichia coli or Salmonella typhimurium indicated that MeBr can be used to disinfect whole nut kernels without resulting in excessive bromide residues, although the MeBr level necessary is higher than that normally used for insect control.

Anti-Bacterial Agents↗
PubMed1983

Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA.

The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.

Amino Acid Sequence↗
PubMed1978

Extracellular water measurements: organ tracer kinetics of bromide and sucrose in rats and man.

Bromide and sucrose distributions were measured as functions of time after tracer injection into 14 rat organs that accounted for 93% of body wt, with the goal of evaluating the use of bromide and sucrose as tracers for the extracellular water volume (ECW). The tracers, Na, 82Br, 125I-labeled human serum albumin, [14C]sucrose, and 3H2O, were used to calculate bromide and sucrose content in red cells, plasma, and 13 blood-free organs. Selective concentration of Br- occurs in RBC, stomach, and skin, accounting in part for the discrepancy between the Br- space and the smaller ECW volume as derived from other methods. Sucrose is rapidly metabolized in the rat and its 14C tracer cannot be used for ECW determination in this species. The kinetics of Br- distribution were estimated in rats and in 16 human subjects by measuring plasma disappearance values and specific organ uptakes. A pharmacokinetic compartmental model was derived, containing explicit parameters for blood flow, diffusion constants, and ECW spaces separately for each organ. Precise fitting of experimental bromide data was achieved for the rat; satisfactory fitting was also achieved in man from more limited plasma and biopsy data.

Animals↗
PubMed1992

Spurious hyperchloremia and decreased anion gap in a patient with dextromethorphan bromide.

Although cold syrup containing dextromethorpan bromide is widely administered, the bromism due to cold syrup has not been reported. We report a patient who had negative anion gap with hyperchloremia and conscious loss because of daily intake of cold complex syrup (containing dextromethorphan bromide 0.4 mg/ml, acetaminophen 8.33 mg/ml) for headache for 4-5 years. The bromide content in cold complex syrup resulted in serum levels of bromide that interfered with the automated analyzers for chloride content. When conscious change is due to bromism, hemodialysis instead of forced hydration and diuresis should be performed immediately. Therefore, patients with a markedly negative anion gap with hyperchloremia should be considered as having halide intoxication.

Bromides↗
PubMed1995

A negative anion gap as a clue to diagnose bromide intoxication.

We report on a patient with bromide intoxication, presenting with confusion, disorientation, and auditory and visual hallucinations after taking a sedative medication containing bromide (mixture menopause; 15 ml containing 1 g potassium bromide) for 1 month. Blood chemistry showed a high chloride level (176 mEq/l) and a negative anion gap (-60 mEq/l). The spurious hyperchloremia was due to interference of chloride ion determination by the ion-selective electrode method with a high level of bromide in serum: 352 mg/dl (44 mEq/l). In this case the only striking abnormality which alerted the physician to the possibility of halide intoxication was the negative anion gap. Hence, a negative anion gap is an important clue which leads to the diagnosis of halide intoxication.

Acid-Base Equilibrium↗
PubMed2005

Measuring the size of the extracellular fluid space using bromide, iohexol, and sodium dilution.

There is a need to find methods to assess the size of the extracellular fluid (ECF) volume without involving radioactive tracers. For this purpose, we applied 3 methods for measuring the ECF volume in 10 male volunteers (mean age, 34 yr). Steady-state plasma bromide concentration (control) was compared to the results of kinetic analysis of plasma iohexol and to kinetic analysis of the dilution of serum sodium after IV infusion of 1 L of isotonic mannitol. The volume of distribution of these tracers was used to indicate the ECF volume. The results disclosed statistically significant correlations between the results of all 3 methods, although the average sodium dilution showed 0.7 L lower values than iohexol and 1.4 L lower than bromide. All three methods correlated significantly with body weight. The percentage of the body weight indicated by the methods was 18.3% (3.1%) for sodium, 19.6% (1.0%) for iohexol, and 20.5% (1.1%) for bromide. We conclude that sodium dilution may be performed at bedside but iohexol and bromide showed less intersubject variability. Iohexol simultaneously measures the glomerular filtration rate and should be a viable clinical option if the hospital performs routine assessments of kidney function using this tracer.

Adult↗
PubMed2005

Biological half-lives of bromide and sodium in the rat are connected and dependent on the physiological state.

The parallel course of the excretion rates of sodium and bromide ions was demonstrated in adult male rats administered simultaneously with 24Na-sodium chloride and 82Br-bromide. These excretion rates were inversely proportional to the magnitude of sodium intake in the animals. The biological half-life of bromide, as a substitute for sodium or chloride, was investigated with the aid of the radionuclide 82Br in animals situated in very different physiological states (i.e., in lactating and nonlactating female rats as well as in young rats of varying ages [2, 4, 6, and 10 wk of age]). The 82Br radioactivity retained in mothers and in whole litters was measured in vivo at appropriate time intervals (up to 240 h) after the application of 82Br-bromide to the mothers. The time-course of the changes in the 82Br radioactivity of the young was calculated as the difference between the rate of 82Br intake in the mother's milk and the 82Br excretion through the kidneys into the urine. The rate of 82Br excretion through the kidneys of the dam could be calculated also. Nonweaned young rats (12 d) had the highest half-life (269 h) and lactating dams had the lowest (44 h). The determined values demonstrated that nonweaned young apparently conserve sodium, because of its relatively low concentration in mother's milk, whereas lactating dams, because of their large food intake, waste sodium.

Aging↗
PubMed1975

Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5.

Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified. Only four of the five fragments theoretically present could be accounted for in major yield when the cyanogen bromide reaction was carried out at room temperature. The NH2-terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent. The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an alpha-chymotryptic peptide. Cleavage at this methionine was increased by 50% when the cyanogen bromide reaction was carried out at 37 degrees. The NH2- and COOH-terminal sequences of five major fragments were determined. The placement of these fragments in the native pepsin molecule was demonstrated. The amino acid sequence of one of the fragments, CB5, was determined. This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37-residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin.

Amino Acid Sequence↗
PubMed1975

Structural studies on rabbit skeletal muscle actin. Ordering of the peptides produced by cleavage with cyanogen bromide.

The 17 peptides produced by cleavage of actin with cyanogen bromide have been ordered with regard to their sequence in the actin molecule. Tryptic digestion of actin followed by isolation of the methionine-containing "overlap" peptides permitted the unique alignment of most, but not all of the cyanogen bromide peptides. However, maleylation of the actin molecule followed by tryptic digestion and isolation of methionine-containing peptides from maleylated actin permitted the proper placement of the remaining cyanogen bromide peptides. The ordering of cyanogen bromide peptides, together with the amino acid sequence of the individual peptides, constitutes the entire amino acid sequence of rabbit skeletal muscle actin (ELZINGA, M., COLLINS, J. H., KUEHL, W. M., and ADENLSTEIN, R. S. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,2687-2691).

Actins↗
PubMed1975

Determination of the bromide space in man by fluorescent excitation analysis of oral bromine.

The bromide dilutional volume determined by intravenous administration of 82Br has been compared with the corresponding volume determined by oral administration of stable bromide in 11 patients with various medical disorders. Stable bromide was assayed by fluorescent excitation analysis using a 109Cd source and a lithium-drifted silicon detector. The average deviation between the fluorescent and the radiobromide dilutional volumes was 4.2% with a standard deviation of +/- 8.5%. This substantiates both the accuracy of the fluorescent excitation method as applied to this tracer and the validity of utilizing oral tracer administration in comparison with intravenous administration. The derived estimates of extracellular fluid volume averaged 28.7% of body weight in the entire group of 11 patients and 25.8% in the 4 normal subjects included in the group. Evaluation of the extracellular fluid space utilizing fluorescent excitation of stable bromide permits high statistical accuracy of sample measurement with great simplicity compared with current chemical methods and with avoidance of the patient radiation exposure associated with 82Br.

Administration, Oral↗
PubMed1975

Bromide partition test in the evaluation of the blood-cerebrospinal barrier in children with congenital hypothyroidism and central nervous system disease.

Serum and cerebrospinal fluid bromide partiton was studied in 11 children with congenital hypothyroidism, in 12 children with tuberculous meningitis, and in 10 children with various neurological disorders. The ratio serum bromide per cerebrospinal fluid bromide (bromide index) was below the critical value of 1.65 in 5 of 11 children with hypothyroidism as compared with 10 of the 12 children with tuberculous meningitis. No clear correlation was found between the test and the clinical and laboratory signs of hypothyroidism. The test is a valuable index of blood-cerebrospinal fluid barrier involvement in hypothyroidism.

Blood-Brain Barrier↗
PubMed1976

Analysis of bromide ion in wine by ion selective electrode.

The use of the bromide ion selective electrode for the determination of bromide ion in wine has been found to be rapid and reliable. The method has been used for still wines and carbonated wines and is applicable to all wines regardless of their country of origin. The method consists of treating a 50 ml aliquot of wine with 2 ml each of 3.75M H3PO4, saturated KNO3, and 1M CuSO4. After 10 min the electrodes are immersed in the samples and a millivolt reading is obtained. One hundred mul 500 ppm bromide ion standard is added and the millivolt reading is taken. Bromide ion concentration in the wine equals (Cdelta x 1)/((antilog deltaE/S)-1) where Cdelta equals 1, deltaE equals the change in potential expressed in millivolts, and S equals the electrode slope.

Bromides↗
PubMed1976

Bromide intoxication.

Bromide intoxication is now an uncommon disease. This report reviews the experience with this disorder at the Johns Hopkins Hospital over the last 23 years and reemphasizes the variable neuropsychiatric symptoms which these patients present. Impaired sensorium, fever, skin rash, abnormal serum chloride, and a history of proprietary drug abuse are clinical features which may suggest the presence of bromide intoxication. Treatment consisting of saline administration often augmented with mercurial, thiazide or "loop" diuretics was uniformly effective. The declining incidence of this disorder is felt to be due to the decrease in physicians' use of prescription bromides and the declining availability of proprietary bromide containing compounds.

Adolescent↗