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The fabrication of artifacts out of glassy carbon and carbon-fiber-reinforced carbon for biomedical applications.

Polymeric carbons are produced by the carbonization of a wide range of organic polymeric systems. We have concentrated on the fabrication of two types of polymeric carbons, glassy carbon and carbon-fiber-reinforced carbon (CFRC), both involving phenolic resin precursors. We describe herein the technology which enables us to make dental implants and heart valves out of glassy carbon. We also show how carbon-fiber-reinforced carbon can be made in the form of rods and plates for orthopedic use and molded before firing to produce complex, rigid, individually sculptured shapes suitable for maxillofacial bone replacement. The mechanical properties will be discussed in relation to the structure of these various forms of polymeric carbon. The main purpose of the work is to show that the technology of polymeric-carbon manufacture is essentially simple and the manufacturing process is readily carried out in laboratories which have already been equipped to fabricate standard dental prostheses.

Acrylonitrile

Carbon dioxide combining properties of the blood of the shore crab Carcinus maenas (L): carbon dioxide solubility coefficient and carbonic acid dissociation constants.

The carbon dioxide solubility coefficient, alphaCO2, and the apparent carbonic acid dissociation constants, K'1 and K'2 were estimated in the serum of the crab Carcinus maenas at various temperatures and ionic strengths. At 15 degrees C, the indirectly determined alphaCO2 value is 0-0499 m-mole l-1 torr-1 for crabs living in normal sea water (salinity ca. 35 percent). It is apparently independent of the serum protein concentration and of the stage of the moulting cycle. For crabs living in undiluted sea water, the mean pK'1 value, determined either gasometrically or titrimetrically, is 6-027 at 15 degrees C. At the same temperature, pK'2=9-29. These values approximate to those of sea water at 35 percent salinity. pK'1 drops as temperature rises; the measured deltapK'1/deltat is -0-0053 pH unit degrees C-1 between 10 and 30 degrees C. PK'1 rises as the ionic strength is lowered. Alignment nomograms have been constructed for the determination of alphaCO2, pK'1 and pK'2 values in relation to various conditions of temperature and salinity.

Animals

Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence.

The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.

Amino Acid Sequence

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.

Arginine

How the rise time of carbon dioxide analysers influences the accuracy of carbon dioxide measurements.

Carbon dioxide measurements are not accurate, especially in children, if the response time of the carbon dioxide analyser is too slow and its output fails to reach the actual carbon dioxide concentration at the end of the breath. The distortion of the carbon dioxide waveform is a function of the "rise time" of the analyser. We have simulated an expired carbon dioxide curve and calculated the rise time required to measure accurately end-tidal carbon dioxide and VCO2 in adults and children. A rise time of 80 ms (10-70%) is sufficient to measure end-tidal carbon dioxide concentration with 5% accuracy in patients with rates of ventilation less than 100 b.p.m. and I:E ratios less than 2:1. We have measured the rise time of 11 commercially available carbon dioxide analysers and found that only six of the 11 responded quickly enough to be accurate for rates up to 100 b.p.m. All 11 responded rapidly enough to measure end-tidal carbon dioxide concentration with 5% accuracy when ventilatory rates were less than 30 b.p.m. To measure VCO2 with 5% accuracy, an analyser should have a rise time of 20 ms. Only one analyser met this specification. An analyser's rise time can be estimated clinically to within 10 (SD 8) ms by a simple breath hold and forced exhalation, thus providing an estimate of the accuracy of carbon dioxide measurements in adults or children.

Anesthesia, Inhalation

Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.

Animals

Direct observation of spin-trapped carbon dioxide radicals in hepatocytes exposed to carbon tetrachloride.

Carbon dioxide radical adducts of the spin trapping agent, alpha-phenyl N-t-butyl nitrone (PBN), have been observed to occur in the urine and bile of rats exposed to carbon tetrachloride as well as in perfusates of liver in which the perfusion medium contained carbon tetrachloride (Connor et al., J. Biol. Chem., 261, 4542, (1986]. The carbon dioxide adduct was proven to be derived from CCl4 by use of 13-C-labelled compound. These adducts were not observed in the liver itself suggesting that they might be rapidly secreted from the liver. However, using isolated hepatocytes, we have demonstrated that the carbon dioxide radical adduct can be observed directly in the liver cells as it is formed. Since this water-soluble adduct cannot be extracted by non-aqueous solvents such as chloroform or toluene, its formation in liver in vivo or in perfused livers was not detected. Lowering the oxygen tension in the system diminished the intensity of production of the carbon dioxide adduct, consistent with the adduct being produced as a result of .OOCCl3 generation. It is not clear the extent to which this adduct is formed as a result of the .CO2 radical or is produced by metabolic oxidation of the trichloromethyl radical adduct of PBN per se to the carbon dioxide radical adduct. The intensity of the signal of the carbon dioxide radical adduct suggests that adduct conversion may be the route of formation since it seems unlikely that a sufficient amount of the halocarbon could be metabolized to .COCl or .CO2 radicals to generate a signal of the magnitude involved.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

End-tidal carbon dioxide concentration, carbon dioxide production, heart rate, and blood pressure as indicators of induced hyperthermia.

In 4 spontaneously breathing, barbiturate-anesthetized dogs, hyperthermia was induced with 2,4-dinitrophenol while rectal temperature, heart rate, mean blood pressure, end-tidal carbon dioxide, and carbon dioxide production (milliliters per minute) were measured continuously. The latter was determined with a pneumotachygraph (to obtain respired volume) and an infrared carbon dioxide analyzer that measured inspired and expired carbon dioxide concentration. Of the five physiologic measurements, the increase in carbon dioxide production preceded the increase in rectal temperature by more than 120 seconds. End-tidal carbon dioxide was an unreliable indicator in the spontaneously breathing animal of approaching hyperthermia during spontaneous breathing due to a transient tachypnea, which decreased end-tidal carbon dioxide. The carbon dioxide production (milliliters per minute) increased immediately and reached three to five times the control value. Blood pressure and heart rate were insensitive indicators of approaching hyperthermia.

2,4-Dinitrophenol

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...

Animals

Calcium carbonate depresses iron bioavailability in rats more than calcium sulfate or sodium carbonate.

Calcium carbonate supplements depress iron bioavailability when consumed with meals. Our objective was to determine whether this effect is due to the calcium, the carbonate or a combination of the two. A rat hemoglobin repletion assay and an in vitro digestion procedure were used to assess the effects of four salts (calcium carbonate, calcium sulfate, sodium carbonate and sodium sulfate) on iron bioavailability. The salts were added to purified rat diets (for the hemoglobin repletion study) or to iron-fortified infant formula (for the in vitro study) at three different levels. Calcium carbonate had the greatest depressive effect on iron bioavailability, depressing hemoglobin iron gain in a dose-related manner. Calcium sulfate and sodium carbonate also depressed hemoglobin iron gain but to a lesser extent and only in rats fed diets containing the highest level of these two salts. Sodium sulfate did not affect hemoglobin iron gain. Significant interactions between cation, anion and salt concentration were found, suggesting that both the cation and the anion in calcium carbonate contribute to the iron absorption-depressing action of this salt. Results from the in vitro experiments were similar to the in vivo results.

Absorption

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species.

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of 13C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.

Acetyl Coenzyme A

Carbon-13 nuclear magnetic resonance studies of adenosylcobalamin and alkylcorrinoids, selectively enriched with carbon-13.

The carbon-13 nuclear magnetic resonance spectra of a series of alkylcorrinoids, selectively enriched with 13C in the alkyl ligand, were recorded at 25.2 MHz and 25 degrees. The nature of the axial ligands markedly affects the chemical shift of the labeled alkyl moiety (trans effect) as well as the 13C resonances of selected carbon atoms of the corrin ring (cis effect). Although a number of factors appear to influence the trans effect on the chemical shift of the alkyl ligand (important among them being electric field effects), the cis effect appears to be dominated by changes in charge density (at the methine bridge carbon atoms, C-5, C-10, C-15) and by steric effects (at the methyl groups at C-1, C-5, and C-15) accompanying axial ligation. Spin-latice relaxation times of several organocorrinoids, selectively labeled with 13C in the ligands attached to cobalt, were also measured. The T1 values of the methylene carbons of [5'-13C]adenosylcobalamin and [2-13C]carboxymethylcobalamin are very similar to that of the methine bridge carbon atom C-10 of the corrin ring, indicating that rotation about the carbon-cobalt bond of these two corrinoids is severely restricted. On the other hand, internal rotation about the carbon-cobalt bond of methylcobalamin is rapid.

Adenosine

Post-translational modification of proteins by 15-carbon and 20-carbon isoprenoids in three mammalian cell lines.

A number of cellular proteins, including p21ras, lamin B, and the G-protein gamma subunits, undergo post-translational modification by 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid moieties derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. In this study, isoprenylated proteins in three mammalian cell lines (Hela cells, Rat-6 fibroblasts and COS cells) were radiolabeled with an isoprenoid precursor, [3H]mevalonate, and resolved by SDS gel electrophoresis. Groups of proteins with different molecular masses were eluted from the gels and the chain-lengths of the radiolabeled isoprenyl groups, released from the proteins by Raney-nickel-catalyzed desulfurization, were established by gel permeation chromatography. 15-Carbon and 20-carbon isoprenyl groups were found in separate classes of proteins within each cell line. With the exception of p21ras, which incorporated a 15-carbon group when expressed in COS cells, the proteins in the region of the 21-28 kDa ras-related GTP binding proteins contained mostly 20-carbon isoprenyl chains. In contrast, proteins belonging to the 66-72 kDa nuclear lamin family, as well as unidentified proteins with molecular masses of 41-46 kDa and 53-55 kDa, contained predominantly 15-carbon isoprenyl chains. The chain-lengths of the isoprenoids associated with particular classes of proteins did not vary from one cell line to another, suggesting that the nature of the isoprenoid modification (farnesyl versus geranylgeranyl) is determined by intrinsic structural features of the proteins, rather than the cell type in which the proteins are expressed.

Animals

The effect of carbonate content and drying temperature on the ESR-spectrum near g = 2 of carbonated calciumapatites synthesized from aqueous media.

The ESR spectrum of X-irradiated carbonated apatites precipitated from aqueous solutions was studied as a function of their carbonate content and drying temperature. When the latter increases from 25 to 400 degrees C, the ESR spectrum is gradually modified and becomes similar to the spectrum of carbonated apatites, synthesized at high temperatures by solid state reactions. The latter ESR spectrum is dominated by CO3(3-)-contributions whereas the spectrum of precipitated samples dried at 25 degrees C can mainly be interpreted in terms of CO2-, CO3-, and O- ions. The behavior of these earlier-reported CO2-, CO3-, and O- centers is now studied as a function of drying temperature. In addition, the Spin Hamiltonian parameters of the CO3(3-) centers are determined and some other new paramagnetic radicals are discussed. It is shown that a CO3(2-) ion at a phosphate lattice site (B-type substitution) may give rise to either a CO2-, CO3-, or CO3(3-) radical on X-irradiation, depending on the sample preparation conditions. A surface CO3(2-) ion may cause a surface CO2-, CO3-, or O- radical. From the reported results it is not unambiguously clear whether the CO3(3-) ion detected in the samples with the relatively lowest carbonate content should be located on the surface or on a hydroxyl lattice site (A-type substitution). An important result is that the absolute concentration of the B-type CO3(3-) ion increases with increasing carbonate content as was also the case for the earlier reported B-type radicals (isotropic CO2- and CO3-). On the other hand, the absolute concentration of the surface radicals decreases with increasing carbonate content. The reported results show that similar deconvolution techniques can be applied in the future for the study of ESR spectra of calcified tissues. This will allow a more efficient phenomenological investigation of the latter.

Apatites

Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms.

Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.

Acetamides

Occupational exposure to carbon monoxide and sulphur dioxide during the manufacture of carbon black.

The manufacture of carbon black is known to generate carbon monoxide and sulphur dioxide in the 'production gas' and the pyrolysis products of the 'production gas', respectively. Adverse health effects have been reported as associated with both contaminants (coronary heart disease with carbon monoxide and respiratory morbidity with sulphur dioxide). A major cross-sectional and longitudinal respiratory morbidity study is being conducted to assess the effects of exposure to carbon black on lung function, on chest X-rays and on responses to a questionnaire. The questionnaire includes questions on respiratory and cardiovascular symptoms, so that information regarding confounding exposure is essential. The working population of 18 manufacturing plants in seven European Countries was split into 13 job title numbers (1-13) which were then amalgamated into five job categories (A-E), with an appropriate (statistically) number of samples taken from each plant-job category. In total, 1322 carbon monoxide samples and 1301 sulphur dioxide samples were taken, using actively pumped longterm colorimetric tubes. In the majority of cases, more than half of the samples in each plant-job category were either zero or trace, thereby preventing the accurate estimation of the average exposure. The highest median carbon monoxide concentration was from the amalgamated data from all 18 plants in job number 9 (furnace operators), the highest median sulphur dioxide concentration was only 'trace'. The large number of zero and trace values also precluded the generation of current and retrospective exposure indices.

Air Pollutants, Occupational

Carbonate dehydratase (carbonic anhydrase) and the fetal lung.

Carbonic anhydrase activity (carbonic dehydratase, EC 4.2.1.1) has been detected in the fetal lungs of stillborn human infants and rhesus monkeys, but a role for this enzyme in the fetal lung has not been elucidated. In utero the mammalian lung develops as a liquid-filled structure, the liquid being secreted by the lung. In the fetal lamb this liquid, when compared with plasma, has a high chloride and a low bicarbonate concentration, suggesting a possible role for carbonate dehydratase. Studies on 10 fetal lambs confirmed the presence of carbonate dehydratase in the lung. Levels at 60-66 days were negligible and rose to 0.30 Meldrum Roughton units/mg protein at about 140 days (term 147 days), with little change after birth. In another six fetal lambs at 135-136 days, inhibition of this enzyme with 100 mg acetazolamide suppressed the mean rate of secretion of lung liquid by 64.5% (P less than 0.005), which correlated with a significant drop in chloride concentration (P less than 0.001). This magnitude of changes in secretion after acetazolamide is of the same order as that occurring in the secretion of cerebrospinal fluid when carbonate dehydratase is inhibited. This observation supports the hypothesis that carbonate dehydratase in fetal lung affects the secretion of lung liquid, although its mechanism is as yet unknown.

Acetazolamide