Metabolism of bromide and its interference with the metabolism of iodine

The present knowledge about the metabolism of bromide with respect to its goitrogenic effects, including some conclusions drawn from our recent research on this subject, is reviewed. Firstly, the biological behavior of bromide ion is compared with that of chloride and iodide. Secondly, the details about distribution and kinetics of bromide ions in the body and in 15 different organs and tissues of the rat are given. Significant correlation between the values of the steady-state concentration of bromide in the respective tissue and of the corresponding biological half-life was found in most tissues examined. A remarkably high concentration of radiobromide was found in the skin, which represents, due to its large mass, the most abundant depot of bromide in the body of the rat. Thirdly, the effects of excessive bromide on the rat thyroid are summarized, along with the interference of exogenous bromide with the whole-body metabolism of iodine. It is suggested that high levels of bromide in the organism of experimental animals can influence their iodine metabolism in two parallel ways: by a decrease in iodide accumulation in the thyroid and skin (and in the mammary glands in lactating dams), and by a rise in iodide excretion by kidneys. By accelerating the renal excretion of iodide, excessive bromide can also influence the pool of exchangeable iodide in the thyroid. Finally, our recent results concerning the influence of high bromide intake in the lactating rat dam on iodine and bromide transfer to the suckling, and the impact of seriously decreased iodine content and increased bromide concentration in mother's milk on the young are discussed. We must state, however, that the virtue of the toxic effects of excessive bromide on the thyroid gland and its interference with the biosynthesis of thyroid hormones, as well as the exact mechanism of bromide interference with postnatal developmental processes remains to be elucidated.     

Studies on the metabolism of rat liver copper-metallothionein.

The degradation of purified 35S-labelled rat liver isometallothioneins (MT) by lysosomal extracts was studied. Zn-MT-I was more readily hydrolysed than Zn-MT-II, but no significant degradation of the Cu-containing metallothioneins could be detected, even after 24 h incubation. The susceptibility of MT to degradation in vitro may be related to the strength of the metal-thiolate bonds. However, the turnover rates of cytosolic MT in vivo, as established by pulse-labelling techniques, are apparently subject to different controls. The half-lives of MT-I and -II in the liver cytosol of Cu2+-injected rats were only 15.4 +/- 1.5 and 18.2 +/- 1.1 h respectively. Approx. 25% of the total liver MT was present in particulate fractions (probably in lysosomes) of the liver and had a half-life of 25.1 +/- 4.1 h.     

A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors.

Several different techniques were used to determine the apparent half-lives of immunoglobulin gamma 2b heavy chain and kappa light chain mRNA's in mouse myeloma 4T001 and a mutant derived from 4T001, i.e., mutant I17. The mutant I17 Ig heavy chain mRNA lacks CH1 and has fused CH2 and CH3 domains resulting in a truncated protein. By all four techniques the Ig heavy chain mRNA from mutant I17 displays a half-life that is approximately 70% the half-life of Ig mRNA in 4T001 cells. However, the absolute values of apparent half-life varied by greater than twofold for both lines among several of the techniques employed. The half-life of Ig gamma 2b mRNA in 4T001 cells was found to be 6.4 h by measuring decay following administration of the adenosine analog DRB to block new mRNA synthesis and 5.7 hr by measuring accumulation in an approach to steady-state labeling protocol. In contrast, the observed Ig mRNA half-lives determined by measuring decay following administration of actinomycin D to block new mRNA synthesis, or in a pulse-chase analysis were 2.9 and 3.8 h, respectively. The apparent half-life for Ig kappa light chain mRNA was the same in the 4T001 and I17 lines using any one technique but the value varied depending on the technique from a high value of 5.9 h following DRB to a low value of 2.4 h with actinomycin decay. Approach to steady-state is theoretically the most accurate method to measure mRNA half-life when that value is less than the doubling time of the cells. Pulse-chase analyses are accurate for measuring mRNA half-life when that value is longer than the effective chase period. Measuring preformed message decay following administration of drugs to block new mRNA synthesis is adaptable over a range of half-lives, but the cells must be shown to retain correct RNA metabolism over the time frame of the experiment. Determining a correct half-life for a particular mRNA may not be feasible using only one method and may, in fact, require several different approaches until a consensus value emerges.     

Determination of bromine and iodine in normal tissues from Beijing healthy adults

The contents of bromine and iodine in samples of heart, liver, spleen, lung, muscle, and hair from healthy adults living in Beijing, China, were determined using epithermal neutron activation analysis. The results indicate that the contents of bromine in lung and iodine in liver are higher than those in other tissues, except human hair. The bromine contents in Beijing human tissues are significantly lower than those in other countries. The contents of iodine are slightly lower than those in other countries, but the difference is not significant. Three biological standard reference materials were simultaneously determined with the samples, and our results agree well with the certified values.     

Bromide kinetics and distribution in the rat. II

The distribution of 82Br-bromide in 15 different organs and tissues of rats has been determined by high-resolution gamma-ray spectrometry and by the scintillation counting technique at different times after the application of Na 82Br, either by subcutaneous injection or by continuous administration in the drinking water. The amount of 82Br-bromide in the various tissues reached its largest uptake within a few hours, and the concentration ratio of 82Br in the tissues to blood remained practically constant between 8 and 396 h after the application. The whole stomach of rats was the only organ of those investigated that had a larger uptake of 82Br than blood. Contrary to some previous findings, the concentration of radiobromide in the thyroid was found not to exceed that in the blood. A remarkably high concentration of 82Br was found in the skin, which represented, because of its large mass, the most abundant depot of bromide in the body of rats. The demonstrated excretion of bromide was mainly renal, at a rate of approximately 5% of the administered dose per 24 h.     

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. Presented in part at the 4th International Symposium on Trace Elements in Human: New Perspectives held in Athens (Greece) on 9–11 October 2003     

Effect of zinc status of rats on the synthesis and degradation of copper-induced metallothioneins.

Injection of Zn2+-adequate and Zn2+-deficient rats with Cu2+ stimulated the incorporation of l-[35S]cysteine into a low-molecular-weight Cu2+-binding protein in both liver and kidney. No significant incorporation of l-[4,5-3H]leucine into this protein occurred, confirming the previous claim that it was metallothionein and not some other leucine-rich protein. The half-life of the protein was found to be 16.9 +/- 1.0 (S.E.)h in the liver of Zn2+-adequate rats but only 12.3 +/- 0.5h in Zn2+-deficient animals. The degradation rate of the metallothionein was similar to the rate of disappearance of Cu2+ and Zn2+ from the protein, indicating that the release of mental from the protein and its catabolism occurred simultaneously. There was no significant difference in the half-lives of the hepatic or renal copper-thioneins in Zn2+-adequate rats.     

Intercellular communication through gap junctions: a potential role in pharmacomechanical coupling and syncytial tissue contraction in vascular smooth muscle isolated from the human corpus cavernosum

Kinetic and steady-state protocols were used to examine the effects of disruption of intercellular communication with heptanol, on contractile responses elicited by activation of the alpha 1-adrenergic receptor in human corporal vascular smooth muscle. For the steady-state studies, strips of corporal tissue from 19 patients were submaximally precontracted with phenylephrine (PE) and then relaxed by the cumulative addition of heptanol. Heptanol completely and reversibly relaxed all tissues studied in a concentration-dependent manner. The heptanol concentration response data were then computer fit to the general logistic equation to obtain pEC50 (negative logarithm of the concentration that elicits one-half of the maximal effect) and slope factor values, with Emax (maximal relaxation) set to 100%. The mean pEC50 and slope factor values, respectively, were 2.86 +/- 0.04 and 1.86 +/- 0.17. Furthermore, kinetic studies on corporal tissues from a subset of the patient population (11 patients) revealed that preincubation of tissues with 2 mM heptanol caused a significant decrease in both the rate and magnitude of PE-induced contractions in all tissues studied, without affecting the rate constant for onset of contraction (k(obs)). The present results on intact tissue extend our previous observations on cultured corporal cells, and support the supposition that intercellular communication through gap junctions may play an important role in the initiation, maintenance and modulation of alpha 1-adrenergic contractions in human vascular smooth muscle.     

Comparison of turnover rates of proteins of the brain, liver and kidney in mouse in vivo following long term labeling.

Intraperitoneal injection of [14C]tyrosine suspension followed by subcutaneous implantation of a [14C]tyrosine pellet in mice produced a fairly constant specific activity of plasma free tyrosine for 5 days, and for 3-5 days in the tissue free amino acid pool. The specific activity of tyrosine in the tissue (brain, liver, and kidney) free amino acid pool was 75-90% of that in plasma. Incorporation of tyrosine into tissue proteins was followed for 5 days in brain; during this time 33% of tissue proteins were labeled. Incorporation for 68 h in liver and kidney showed labeling of over 70% of the protein of these tissues. These percentages assume a homogeneous tissue free tyrosine pool as the precursor. The rate of incorporation initially was 0.6, 2.8, and 2.0% per h in brain, liver, and kidney protein, respectively. These rates decreased in longer term experiments. The best fit to the incorporation curves was obtained by assuming the following average half-lives for tissue proteins: brain, two compartments, 5.7% with a half-life of 15 h, 94.3% with a half-life of 10 days; liver, a single compartment with a 26-h half-life; kidney, two compartments, 41% with an 18-h half-life, and 59% with a 63-h half-life.     

Simultaneous 280 MHz EPR imaging of rat organs during nitroxide free radical clearance.

A radio frequency (RF) (280 MHz) electron paramagnetic resonance (EPR) spectroscopy and imaging apparatus has been used to localize a pyrrolidine nitroxide free radical in the rat abdomen and thorax. The nitroxide 2,2.5.5,-tetramethylpyrrolidine-1-oxyl-3- carboxylic acid (PCA) had a whole body monoexponential decay with half-life of 13.3 +/- 0.7 (n = 4), 19.4 +/- 0.2 (n = 3), and 23 +/- 2 (n = 6) min for 1, 2, and 3 mmol/kg PCA, respectively. Up to seven one-dimensional longitudinal projections were collected on six rats in the presence of a 8 mT/m field gradient. With an injection dose of 3 mmol/kg, PCA half-lives were 19 +/- 1, 17 +/- 2, and 22 +/- 2 min (n = 6) in the lower abdomen, in the liver, and in the thorax, respectively. Thorax half-life was significantly longer than liver half-life. Sequential two-dimensional images of PCA distribution in a plane longitudinal to the rat body were obtained from eight spectra in the presence of a gradient of 12 mT/m (acquisition time 5 min; spatial resolution 8 mm). After 7 min, the nitroxide was detectable in the left side of the thorax area, but it was mostly localized in the liver. PCA was more uniformly distributed in the image collected after 17 min.     

Protein degradation in the mouse blastocyst.

The degradation characteristics of 56 individual newly synthesized proteins of the Day 4 mouse blastocyst have been examined employing double isotope labeling of proteins for half-life measurement and two-dimensional electrophoresis for separation of proteins. The half-lives ranged from 1 to approximately 30 h with a mean of 12.4 h. Several proteins appeared to have half-lives greater than 30 h but decay times were insufficient to provide precise information for these proteins. The results suggest there is a tendency for proteins with acidic isoelectric points to be degraded more rapidly than basic proteins, and for high molecular weight proteins to be degraded more rapidly than low molecular weight proteins. Although the regressions of these two parameters on half-life were not significant, the direction and magnitude of the trends were similar to those previously described for liver proteins. Two specific proteins, tubulin and actin, were tentatively identified, and their half-lives determined. Tubulin had a half-life of 9.0 h. The half-lives of the provisionally identified gamma, beta, and alpha forms of actin were 2.2, 8.7, and 5.4 h respectively.     

A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10(5 )deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 +/- 0.002 for liver to 0.015 +/- 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of gamma-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after gamma-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.     

Biological half-life of bromide in the rat depends primarily on the magnitude of sodium intake

The parallel course of the excretion rates of bromide and sodium ions was demonstrated in adult male and female rats administered simultaneously with potassium 82Br-bromide and 24Na-sodium chloride. The animals were exposed to various intakes of sodium ions accompanied with five different anions: Br-, Cl-, HCO3-, ClO4-, and SCN-. Regardless of the anion accompanying the sodium ion, the excretion rates of 82Br- and 24Na+ ions were proportional to the magnitude of sodium intake in the animals. Hence, we have proved our hypothesis that the biological half-life of bromide depends on the magnitude of sodium intake rather than on the intake of chloride.     

Enantioselective pharmacokinetics of ethotoin in humans following single oral doses of the racemate.

Racemic ethotoin (1000 mg) was administered orally as a single dose to six healthy adult volunteers. Blood samples were collected at appropriate times for 120 h following the dose. Ethotoin was quantified enantio-selectively in plasma using a novel chiral column HPLC procedure. One of the enantiomers of the chiral metabolite, 5-phenylhydantoin, was also quantified in the HPLC method. The Cmax and AUC0-infinity values for (+)-(S)-ethotoin were significantly greater than those for (-)-(R)-ethotoin (ratio of mean AUC0-infinity values 0.88), but the elimination half-lives of the isomers were virtually identical [12.35 +/- 5.15 h for (-)-(R)-ethotoin; 12.28 +/- 5.34 h for (+)-(S)-ethotoin]. Parameters derived from AUC0-infinity (Cl0/F and V(area)/F) also differed slightly between the isomers. The data were interpreted as indicating a small difference in the absorption of the two isomers; it seemed unlikely, in terms of the identical elimination rates, that their metabolic profiles would differ greatly. The 5-phenylhydantoin was eliminated with a significantly longer half-life (18.69 +/- 6.11 h) than that of ethotoin. Enantioselectivity in the pharmacokinetics of ethotoin is therefore a minor issue.     

Interaction of bromine with iodine in the rat thyroid gland at enhanced bromide intake

In experiments with rats, we have found that at enhanced intake of bromide, bromine does not replace chlorine in the thyroid; it replaces iodine. Under our experimental conditions, more than onethird of the iodine content in the thyroid was replaced by bromine. In the thyroid, bromine probably remained in the form of bromide and, in proportional to its increased concentration, the production of iodinated thyronines decreased, with the sum of the iodine and bromine concentrations being constant at the value of 20.51±1.16 μmol/g dry wt of the thyroid. In contrast to other organs, the biological behavior of bromine in the thyroid is not similar to the biological behavior of chlorine but resembles more that of iodine.     

Mechanism of hepatic assimilation of dipeptides. Transport versus hydrolysis

To investigate dipeptide assimilation by the liver, a series of interrelated experiments were performed in rats. Partial hepatectomy prolonged the plasma half-life (min) of Gly-Ala (3.42 +/- 0.22 versus 4.90 +/- 0.35, p less than 0.05) but had no significant effect on plasma half-life of Gly-Leu, Gly-Pro, or Gly-Sar. We then investigated the rate of disappearance (mumol X (g liver X h)-1) of the above four dipeptides (initial concentration = 1 mM) from the medium during isolated liver perfusion. The order of dipeptide disappearance was: Gly-Leu (8.75 +/- 0.65) greater than Gly-Ala (3.36 +/- 0.46) greater than Gly-Pro (1.29 +/- 0.54) greater than Gly-Sar (0.35 +/- 0.12). This order of dipeptide disappearance corresponded exactly to the order of the rates of glycine accumulation in the medium during liver perfusion with the four dipeptides. Addition of glucagon had no effect on the disappearance rate of Gly-Ala from the medium, but reduced accumulation rates of glycine (3.39 +/- 0.30 versus 1.42 +/- 30, p less than 0.01) and alanine (4.42 +/- 0.66 versus 1.35 +/- 0.39, p less than 0.01). Finally, we found that hydrolysis by the liver plasma membranes and/or perfusion medium accounted for disappearance of dipeptides. In conclusion, the liver does not appear to have a transport system for dipeptides, but assimilates dipeptides by extracellular hydrolysis. Hydrolysis is achieved by enzymes either located on the plasma membranes or released from the cytosol. The amino acid residues released as the result of dipeptide hydrolysis are then taken up by the liver.     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

Glycosylation accelerates albumin degradation in normal and diabetic dogs

Nonenzymatic glycosylation of albumin was associated with an increased catabolic rate and decreased protein half-life in both normal and diabetic animals. The fractional catabolic rate of glycosylated albumin was increased significantly over albumin, from 0.100 +/- 0.004/day to 0.131 +/- 0.007/day in normal animals and from 0.104 +/- 0.004/day to 0.138 +/- 0.007/day when these animals were made diabetic with alloxan. The half-lives of Alb and GlyAlb in normal dogs were 6.81 +/- 0.12 days and 4.97 +/- 0.21 days, respectively. In diabetic animals, the half-lives of Alb and GlyAlb were 7.48 +/- 0.21 and 5.21 +/- 0.24 days, respectively. The increased catabolism of GlyAlb may reflect chronic increased extravasation of glycosylated plasma proteins, which are known to be increased in diabetes, into the microvascular wall.     

Adjustment of phenprocoumon dosage utilizing a pharmacokinetic model

According to a pharmacokinetic model, the adjustment of a phenprocoumon (PPC) standard dosage based on experimentally determined means values of the parameters volume of distribution and biological half-life will yield in more than 50% of the individuals the desired plasma PPC concentration. In 25 patients it was tested, whether the thus derived loading and maintenance doses, 376.8 and 35.2 micrograms PPC per kg b. wt., resp., actually lead to the predicted optimum plasma PPC concentration of 2.0 micrograms/ml. After initiating dosage, the plasma PPC concentrations were determined over a time period of 30 days. As predicted by the model, in 72% of the patients the average steady-state plasma PPC concentrations were within the range of 1.7 and 2.3 micrograms/ml. The data obtained were used to newly calculate mean values of the volume of distribution, the biological half-life, and the total body clearance of PPC. The mean biological half-life of PPC derived was somewhat shorter than that used for the calculation of the standard dose. Quick-values were estimated concomitantly with the plasma PPC concentrations. They revealed an optimum anticoagulation (15-25%) in 96% of the patients.     

Simultaneous In Vivb Monitoring of Cerebral Deoxyglucose and Deoxyglucos-6-Phosphate by l3C{lH} Nuclear Magnetic Resonance Spectroscopy

The capacity of brain to dephosphorylate glucose-6-phosphate has been established, but the magnitude and significance of this capacity in vivo are debated, particularly in regard to dephosphorylation of the glucose analog 2-deoxyglucose. We now report results of external measurement in the brains of conscious rats with simultaneous resolution and quantification of both 2-deoxyglucose and its phosphorylated product by nuclear magnetic resonance (NMR) techniques that used 2-[6-13]deoxyglucose together with proton-decoupled 13C surface-coil spectroscopy. As NMR techniques require large doses of 2-deoxyglucose, a dose comparison was first made using decay curves of total label after tracer doses of 2-[14C]deoxyglucose without versus with unlabeled deoxyglucose at 500 mg/kg (the NMR dose). Similar cerebral half-lives for the two doses were found, and no behavioral evidence for toxicity of the NMR dose was seen. In vivo NMR monitoring of conscious rats showed that the analog reached maximal cerebral concentration within 10 min of the intravenous bolus and decayed with a half-life of 29 +/- 7 min (n = 4; mean +/- SEM), whereas 2-deoxyglucose-6-phosphate reached peak concentration between 30 and 40 min and decayed with a half-life of 2.1 +/- 0.3 h, equivalent to a fractional loss of 0.8%/min. Thirty-one percent (+/- 5%) of the total analog pool (which showed a half-life of 1.4 h) consisted of 2-deoxyglucose at 45 min after the bolus. The results support an active but limited role for dephosphorylation by normal brain in glucose analog (and potentially glucose) metabolism in the unstimulated conscious rat and a wide concentration range for the metabolic operations involved.