Old and new approaches to the interpretation of acid-base metabolism, starting from historical data applied to diabetic acidosis.

The approach to acid-base chemistry in medicine includes several methods. Currently, the two most popular procedures are derived from Stewart's studies and from the bicarbonate/BE-based classical formulation. Another method, unfortunately little known, follows the Kildeberg theory applied to acid-base titration. By using the data produced by Dana Atchley in 1933, regarding electrolytes and blood gas analysis applied to diabetes, we compared the three aforementioned methods, in order to highlight their strengths and their weaknesses. The results obtained, by reprocessing the data of Atchley, have shown that Kildeberg's approach, unlike the other two methods, is consistent, rational and complete for describing the organ-physiological behavior of the hydrogen ion turnover in human organism. In contrast, the data obtained using the Stewart approach and the bicarbonate-based classical formulation are misleading and fail to specify which organs or systems are involved in causing or maintaining the diabetic acidosis. Stewart's approach, despite being considered 'quantitative', does not propose in any way the concept of 'an amount of acid' and becomes even more confusing, because it is not clear how to distinguish between 'strong' and 'weak' ions. As for Stewart's approach, the classical method makes no distinction between hydrogen ions managed by the intermediate metabolism and hydroxyl ions handled by the kidney, but, at least, it is based on the concept of titration (base-excess) and indirectly defines the concept of 'an amount of acid'. In conclusion, only Kildeberg's approach offers a complete understanding of the causes and remedies against any type of acid-base disturbance.

Human granulocyte generation of hydroxyl radical.

Human granulocytes were capable of oxidizing 2-keto-4 thiomethylbutyric acid to ethylene during phagocytosis or membrane perturbation. The reaction required hydrogen peroxide and superoxide and in addition was inhibited by various hydroxyl radical (OH) scavengers. These observations represent direct evidence for the generation of OH by human granulocytes. Further, inhibition of ethylene generation by azide and cyanide suggests that OH generation in granulocytes may be linked to myeloperoxidase.

Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy.

Electron spin resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed to detect the formation of hydroxyl radicals (OH.) by phagocytosing polymorphonuclear leukocytes (PMN). An electron spin resonance signal with the identical g value and splitting characteristics of the DMPO/OH). adduct was detected on incubation of normal PMN with opsonized zymosan. Adduct formation was strongly inhibited by superoxide dismutase and by the OH. scavenger mannitol, but catalase had little or no effect. (DMPO/OH). was not formed by PMN from a patient with chronic granulomatous disease; in contrast, adduct formation by PMN which lack myeloperoxidase was greater than normal. These findings are discussed in relation to the formation of OH. by PMN.

Spontaneous oxygen radical generation by sickle erythrocytes.

Since the various membrane abnormalities of sickle erythrocytes might result from excessive accumulation of oxidant damage, we have measured the generation of superoxide, peroxide, and hydroxyl radical by normal and sickle erythrocytes using assays involving reduction of cytochrome c, aminotriazole inhibition of catalase, and methane evolution from dimethyl sulfoxide, respectively. Compared with normal erythrocytes, sickle erythrocytes spontaneously generate approximately twice as much superoxide, peroxide, and hydroxyl radical. One possible source of hydroxyl radical generation was identified as hemichrome, excessive amounts of which are bound to sickle erythrocyte membranes. Hemichrome did not generate hydroxyl radical when exposed to superoxide alone or peroxide alone. However, in the presence of both superoxide and peroxide, hemichrome greatly facilitated hydroxyl radical generation. Supporting this, normal erythrocyte membranes induced to acquire sickle hemichrome concomitantly acquired an enhanced ability to mediate hydroxyl radical generation. Finally, sickle erythrocyte membranes greatly enhanced superoxide/peroxide-driven hydroxyl radical generation as compared with normal erythrocyte membranes. These data suggest that an excessive accumulation of oxidant damage in sickle erythrocyte membranes might contribute to the accelerated membrane senescence of these cells. They further indicate that accumulation of oxidant damage could be a determinant of normal erythrocyte membrane senescence.

Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical.

The ability of intact human red cells to scavenge extracellularly generated H2O2 and O2-, and to prevent formation of hydroxyl radicals and hypochlorous acid has been examined. Red cells inhibited oxidation of ferrocytochrome c by H2O2. Cells treated with aminotriazole no longer inhibited, indicating that protection was almost entirely due to intracellular catalase. Contribution by the GSH system was slight, and apparent only with low H2O2 concentrations when catalase was inhibited by aminotriazole. The cells were about a quarter as efficient at inhibiting cytochrome c oxidation as an equivalent concentration of purified catalase. No inhibition of O2(-)-dependent reduction of ferricytochrome c or nitroblue tetrazolium was observed, although extracted red cell superoxide dismutase inhibited nitroblue tetrazolium reduction at one fortieth the concentration of that in the cells. Red cells efficiently inhibited deoxyribose oxidation by hydroxyl radicals generated from H2O2, O2- and Fe(EDTA), and myeloperoxidase-dependent oxidation of methionine to methionine sulfoxide by stimulated neutrophils. Most of the red cell inhibition of hydroxyl radical production, and all the inhibition of methionine oxidation, was prevented by blocking intracellular catalase with aminotriazole. Thus red cells are able to efficiently scavenge H2O2, but not O2-, produced in their environment, and to inhibit formation of hydroxyl radicals and hypochlorous acid. They may therefore have an important role in extracellular antioxidant defense.

Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea.

Toxic, partially reduced metabolites of oxygen (toxic oxygen radicals) are increasingly implicated in acute leukocyte-mediated tissue injury. To further probe the roles of oxygen radicals in acute lung edema, I studied the effects of a recently described and very potent oxygen radical scavenger, dimethylthiourea (DMTU) (Fox, R. B., R. N. Harada, R. M. Tate, and J. E. Repine, 1983, J. Appl. Physiol., 55:1456-1459) on polymorphonuclear leukocyte (PMN) oxidant function and on two types of lung injury mediated by oxygen radicals and PMN. DMTU (10 mM) blocked 79% of hydroxyl radical (OH) production by PMN in vitro without interfering with other PMN functions, such as O-2 production, myeloperoxidase activity, chemotaxis, degranulation, or aggregation. When isolated rat lung preparations were perfused with PMN activated to produce OH, lung weights were increased from 2.3 +/- 0.2 to 11.2 +/- 0.8 g. DMTU (10 mM) prevented 70% of these increases (lung weights, 5.0 +/- 1.1 g, P less than 0.005). Finally, when intact rats were exposed to 100% O2 for 66 h, lung weight:body weight ratios were increased from 5.78 +/- 0.33 to 8.87 +/- 0.16 g. DMTU (500 mg/kg) prevented 83% of this hyperoxia-induced lung edema in vivo (lung:body weight ratios, 6.05 +/- 0.21, P less than 0.001). Pharmacokinetic studies showed that DMTU diffused effectively into lung interstitial fluids and had a relatively long half-life (25-35 h) in the circulation. Because a variety of oxygen radicals, such as superoxide (O-2), hydrogen peroxide (H2O2), or OH are produced by PMN, there is usually some uncertainty about which one is responsible for injury. However, in these studies, DMTU did not scavenge O-2 and scavenged H2O2 only very slowly while scavenging OH very effectively. Therefore, DMTU may be useful in the investigation of the roles of oxygen radicals, especially OH, in acute granulocyte-mediated tissue injury.

Metal-ion-directed site-specificity of hydroxyl radical detection.

A wide variety of .OH detectors are in use for determination of biological .OH production. The chemical generation of .OH is site-specific with respect to the metal-binding site, and thus .OH detectors with metal-binding properties may affect the biological damage and bias .OH detection. The present study shows that both salicylate and phenylalanine, added as low molecular weight .OH indicators, decreased Cu(II) binding to erythrocyte ghosts. In a cell-free system, Cu(II) complexed to both salicylate and phenylalanine. Phenylalanine is a stronger Cu(II) chelator than salicylate, both when competing for Cu(II) bound to ghosts and when competing directly with each other. When OH radicals were generated by ascorbate and Cu(II), the amount of .OH detected as dihydroxybenzoates was proportional to the amount of .OH produced. However, when phenylalanine was added to this system, the efficiency of .OH detection by salicylate strongly decreased, concomitant with the transfer of Cu(II) binding from salicylate to the amino acid. This decrease was larger than that predicted by calculations for random competition of the two detectors for .OH. Deoxyribose and mannitol, which do not bind copper appreciably, competed poorly with salicylate for the .OH. Hydroxylation of phenylalanine, on the other hand, was only slightly affected by the presence of salicylate and unaffected by deoxyribose and mannitol. These results suggest that the detection of .OH by low molecular weight .OH indicators was related to the relative affinity of the detectors for the catalyzing metal, and thus partially site-specific. Furthermore, glutamate, which does not contain an aromatic ring but binds Cu(II) with considerable affinity, competed strongly with salicylate for the .OH, indicating that metal-binding properties rather than the presence of an aromatic ring were the cause of the deviation from random competition. The results indicate that .OH indicators with metal-binding properties affect the distribution of catalytic metal ions in a biological system, causing a shift of free radical damage and localizing a site-specific reaction of .OH on these detectors, with a resulting positive bias in the apparent .OH production.

Proton (or hydroxide) fluxes and the biphasic osmotic response of human red blood cells.

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).

Do humans neutrophils form hydroxyl radical? Evaluation of an unresolved controversy.

Hydroxyl radical is a potent oxidizing agent of potential importance in human pathobiology. Since neutrophilic phagocytes make superoxide and hydrogen peroxide during phagocytosis, it has been proposed that hydroxyl radical is also formed. In this paper we review the literature which supports or refutes formation of hydroxyl radical by neutrophils and the mechanism(s) by which this radical might be formed. We conclude that there is no definitive proof for hydroxyl radical formation by neutrophils. In fact, neutrophil release of lactoferrin and myeloperoxidase appears to limit formation of this radical. Future studies are likely to determine whether superoxide released by neutrophils interacts with target substrates to allow formation of hydroxyl radical.

Conversion of superoxide generated by polymorphonuclear leukocytes to hydroxyl radical: a direct spectrophotometric detection system based on degradation of deoxyribose.

Hydroxyl radical is produced secondarily after phagocytic cells have been stimulated to generate superoxide anion. The systems used most commonly for detection of cell-generated hydroxyl radical are often inconvenient for routine biomedical research. We have modified an assay used heretofore in cell-free systems, that is, the degradation of deoxyribose, and adapted it for use with neutrophils. The time and dose responses of the system, requirement for chelated iron, inhibition profiles with various scavengers, and correlation with superoxide production have been ascertained. The method correlated strongly with a standard but more cumbersome technique. Values for a normal population are provided. The method can readily be used to study the parameters of superoxide-hydroxyl radical conversion by cells in various disease or treatment states.

The production of hydroxyl radicals by adriamycin in red blood cells.

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.

Macroxyproteinase (M.O.P.): a 670 kDa proteinase complex that degrades oxidatively denatured proteins in red blood cells.

Erythrocytes and reticulocytes are shown to undergo rapid rates of protein degradation following exposure to oxidative stress. Experiments with ATP depletion revealed that, unlike the proteolysis of many other abnormal proteins, the degradation of oxidatively modified proteins is an ATP-independent process. Ion exchange chromatography (DEAE Sepharose CL-6B), ammonium sulfate precipitation, gel filtration chromatography (Sephacryl S-300 or Sepharose CL-6B), and a second ion exchange step were used to resolve the activity responsible for degrading oxidatively modified proteins from (dialyzed) cell-free extracts of erythrocytes and reticulocytes. Gel filtration studies revealed that some 70-80% of the activity in erythrocytes, and some 60-70% of the activity in reticulocytes, is expressed by a 670 kDa proteinase complex that is not stimulated by ATP (in fact, ATP is slightly inhibitory). This proteinase complex is inhibited by sulfhydryl reagents, serine reagents, and transition metal chelators, and has a pH optimum of 7.8. We propose the trivial name "macroxyproteinase" or "M.O.P." (abbreviated from Macro-Oxy-Proteinase) for the complex because of its large size, substrate preference (oxidatively modified proteins), and inhibitor profile (which indicates multiple catalytic sites). Electrophoresis studies of the 670 kDa M.O.P. complex revealed the presence of 8 distinct polypeptide subunits with the following apparent molecular sizes: 21.5, 25.3, 26.2, 28.1, 30.0, 31.9, 33.3, and 35.7 kDa. The large molecular size of the M.O.P. complex, its ATP- and ubiquitin-independence, its inhibitor profile, its distinctive subunit banding pattern in denaturing electrophoresis gels, its pH optimum, and its proteolytic profile with fluorogenic peptide substrates all indicate that M.O.P. is identical to 600-700 kDa neutral/alkaline proteinase complexes that have been isolated from a wide variety of eucaryotic cells and tissues, but for which no function has previously been clear. We propose that macroxyproteinase is responsible for catalyzing most of the selective degradation of oxidatively denatured proteins in red blood cells. We further suggest that M.O.P. may perform the same function in other eucaryotic cells and tissues.

Myeloperoxidase oxidation of sulfur-centered and benzoic acid hydroxyl radical scavengers.

Myeloperoxidase (MPO) oxidizes sulfur-centered and benzoate hydroxyl radical scavengers through formation of HOCl. Sulfur-centered hydroxyl radical scavengers compete with benzoate as antioxidants of HOCl. We conclude from these observations that competition experiments between benzoate and sulfur-centered hydroxyl radical scavengers are not sufficiently specific to infer participation of hydroxyl radicals in oxidative reactions mediated by neutrophils because of the unique action of MPO in affecting oxidation of the test radical scavengers.

Measurement of oxidizing radicals by polymorphonuclear leukocytes.

O2 radicals are important in health and disease. The most commonly used ways of identifying O2 radicals in PMN are described above. Several shortcomings exist in these methods reflecting the unusual, complex nature of O2 radical biochemistry. Some general principles include (1) O2 radicals are very short-lived, reacting with many other compounds and each other quickly. (2) There are no highly specific assays for O2 radicals. (3) Highly specific scavengers of O2 radicals also do not exist. (4) No methods have been found to detect and quantitate O2 radicals in vivo. (5) Solubility and membrane permeability of various scavengers and/or test reagents may affect the measurement of O2 radicals in PMN and other biological systems. In general, the best approach to measurement of O2 radicals involves using the best assay available and showing that the reaction is inhibited by scavengers in proportion to their reactivity with the specific O2 radical being assayed.

Defective oxidative metabolism in newborn neutrophils: discrepancy between superoxide anion and hydroxyl radical generation.

Investigation of oxidative metabolism in neutrophils (PMNs) from newborns was performed by measuring generation of superoxide anion (.O2-) and production of hydroxyl radical (.OH) in the resting state and after stimulation with opsonized zymosan or phorbol myristate acetate (PMA). Neutrophils from cord blood of ten term infants and normal adult controls were tested simultaneously. Cord PMNs generated significantly more .O2- than paired adult controls when stimulated with opsonized zymosan (P less than .01) and produced less .OH with PMA (P less than .005). When the amount of .O2- and .OH released by newborn PMNs with both stimuli was expressed as percent of values obtained from paired adult controls, there was a discrepancy in the generation of these two radicals: newborn PMNs produced relatively less .OH compared to .O2-. This decreased ability to produce .OH could underlie defective bactericidal activity in PMNs of neonates.

Hydrogen peroxide-induced glutathione depletion and aldehyde dehydrogenase inhibition in erythrocytes.

To study relationships between lipid peroxidation and aldehyde dehydrogenase (ALDH) inhibition, the Stocks and Dormandy model of H2O2-induced lipid peroxidation in erythrocytes was employed. Hydrogen peroxide treatment of erythrocytes and erythrocyte lysates caused a dose-dependent inhibition and depletion of ALDH and reduced glutathione (GSH) respectively. Complete ALDH inhibition and glutathione depletion occurred before significant lipid peroxidation was detected by HPLC analysis of malondialdehyde-thiobarbituric acid adducts. Hydroxyl radical scavengers did not antagonize the hydrogen peroxide-induced enzyme inhibition. Studies with the iron chelator desferrioxamine suggested that the hydrogen peroxide-induced ALDH inhibition was mediated by iron in erythrocyte lysates but not in semi-purified (and Chelex-treated) ALDH preparations. Glutathione peroxidase reduction of H2O2 exhibited an anomalous GSH dependence which was not in agreement with the accepted reaction mechanism. Reduced glutathione also antagonized the hydrogen peroxide-induced ALDH inhibition by possible complex formation with the enzyme. A hypothetical model is presented which accounts for the observed responses to hydrogen peroxide.

Insulin-stimulated conversion of D-[5-3H] glucose to 3HOH in the perifused isolated rat adipocyte.

Characteristics of basal and insulin-stimulated glucose utilization by perifused adipocytes have been investigated by measuring the formation of 3HOH from D-(5-3H) glucose. At a glucose concentration of 0.55 mmol/L, basal glucose utilization ranged from 0.5 to 1.0 nmol/min/10(6) cells. Perifused adipocytes showed a maximal response to insulin of a threefold to fourfold increase in the conversion of (5-3H) glucose to 3HOH with a half-maximal response at an insulin concentration of 20 microU/mL. The response to insulin was blocked by phlorizin and cytochalasin B, competitive inhibitors of glucose transport, consistent with an effect of insulin on glucose transport. Insulin increased the Vmax for glucose metabolism but had no effect on the apparent affinity for glucose utilization. The characteristics of glucose utilization and the stimulation of glucose metabolism by insulin in the perifused adipocyte are therefore similar to characteristics previously observed with incubated adipocytes. Because insulin can readily be removed from the system, perifused adipocytes are especially suited for studying the termination of insulin action. The termination of insulin-stimulated glucose metabolism occurred at the same rate in the presence of tracer (1 nmol/L) (5-3H)-glucose alone as when 0.55 mmol/L glucose or 2 mmol/L pyruvate were added to the perifusion buffer. The halftime for this process in both cases was approximately 40 minutes. These data suggest that the presence of metabolizable substrate is not required for the termination of the insulin response, but the time course suggests that termination requires more than simply insulin-receptor dissociation.

Effect of diethyldithiocarbamate on diabetogenic action of alloxan in rats.

To determine whether alloxan action is mediated by hydroxyl radicals in vivo, we assayed methane sulfinic acid (MSA), a product of the trapping reaction of dimethyl sulfoxide (DMSO) with hydroxyl radicals. In DMSO-treated rats, the plasma levels of MSA were increased after injection of alloxan (75 mg/kg). This supports the hypothesis that the diabetogenic action of alloxan is mediated by hydroxyl radicals in vivo. The role of cytosolic superoxide dismutase (SOD) in protecting B cells against chemically induced diabetes was studied in rats injected intraperitoneally with diethyldithiocarbamate (DDC). When rats were injected intraperitoneally with DDC (750 mg/kg), the SOD activity at 2.5 h was decreased by 44% in the whole pancreas. The decreased SOD activity was affected by DDC but not by alloxan. Intraperitoneal injection of rats with DDC (750 mg/kg) increased diabetogenic susceptibility to a nondiabetogenic dose of alloxan (20 mg/kg). Subcutaneous injection of vitamin E, prior to administration of both DDC and alloxan, provided partial protection to the rats against the diabetogenic action. These findings suggest that the susceptibility to diabetogenic action of alloxan in B cells is augmented when the cellular SOD activity is inhibited. Thus, cellular SOD may play an important role in the maintenance of B cell function.

Effect of sodium/potassium salt (potash) on the bioavailability of ibuprofen in healthy human volunteers.

The influence of sodium/potassium salt water extract incorporated in a traditional meal on the bioavailability of Ibuprofen tablets 400mg dose was studied in 6 healthy human volunteers. There was a statistically significant decrease in the plasma levels of ibuprofen, and its metabolites, hydroxy-ibuprofen and carboxy-ibuprofen, respectively, when the meal containing sodium/potassium salt extract was administered with the ibuprofen tablets than when taken under fasting state or with the meal without the fruit extract. The Cmax, AUC0-6hr and Ka for ibuprofen decreased from 38.04 +/- 0.70microg/ml to 20.06 +/- 1.21microg/ml (p<0.05); 28.030 +/- 2.40microg/ml.hr to 14.180 +/- 1.12microg/ml.hr (p<0.05) and 1.048 +/- 0.02hr(-1) to 0.602 +/- 0.03hr(-1). Similarly, the Cmax for hydroxy-ibuprofen and carboxy-ibuprofen decreases from 43.04 +/- 0.76microg/ml to 27.21 +/- 0.24microg/ml (p<0.05) and 48 +/- 0.71microg/ml to 31.08 +/- 0.12microg/ml (p<0.05) respectively; while AUC0-6hr for hydroxy-ibuprofen decreased from 34.120 +/- 0.49microg/ml.hr to 16.410 +/- 0.27microg/ml.hr while that of carboxy-ibuprofen decreased from 36.121 +/- 1.97microg/ml.hr to 19.278 +/- 0.92microg/ml.hr respectively. The Kel for hydroxy-ibuprofen increased from 0.71 +/- 0.94 hr(-1) to 0.81 +/- 0.21 hr(-1) (p<0.05) respectively. The study has indicated that sodium/potassium salt extract significantly decreased the bioavailability of ibuprofen.

Ceria-engineered nanomaterial distribution in, and clearance from, blood: size matters.

AIMS: Characterize different sized ceria-engineered nanomaterial (ENM) distribution in, and clearance from, blood (compared to the cerium ion) following intravenous infusion. MATERIALS & METHODS: Cerium (Ce) was quantified in whole blood, serum and clot (the formed elements) up to 720 h. RESULTS: Traditional pharmacokinetic modeling showed best fit for 5 nm ceria ENM and the cerium ion. Ceria ENMs larger than 5 nm were rapidly cleared from blood. After initially declining, whole blood 15 and 30 nm ceria increased (results that have not been well-described by traditional pharmacokinetic modeling). The cerium ion and 5 and 55 nm ceria did not preferentially distribute into serum or clot, a mixture of cubic and rod shaped ceria was predominantly in the clot, and 15 and 30 nm ceria migrated into the clot over 4 h. CONCLUSION: Reticuloendothelial organs may not readily recognize five nm ceria. Increased ceria distribution into the clot over time may be due to opsonization. Traditional pharmacokinetic analysis was not very informative. Ceria ENM pharmacokinetics are quite different from the cerium ion.