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Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact sites between the two organelles. Furthermore, the presence of this glycoprotein in at least two major mitochondrial multienzyme complexes would be consistent with a role in mitochondrial translocations.

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A method for the isolation of highly purified peroxisomes from guinea pig small intestine was developed. This two-stage process involved a rate-dependent banding of a light-mitochondria lambda-fraction followed by a density-dependent banding of the catalase enriched fractions obtained from the first step, using a horizontal rotor. Furthermore, the subcellular localization of glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in guinea pig small intestine was examined. Analysis of density-gradient fractions indicated that approximately 3-4% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and that it is localized to the matrix of peroxisomes. It is therefore suggested that a peroxisomal source of NADPH may be utilized by enzyme systems that use NADPH specifically as a reductant.

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The drug release and intratumoral residence time of small and large liposomes composed of saturated phospholipids and cholesterol were measured in vitro and in tumor-bearing rats by computed tomography. The in vitro release of metrizamide at 37 degrees C was higher in tissue fluid than in diluted serum and PBS-buffer. The extent of release was 20%/48 h for the 100 nm-liposomes and 10%/48 h for the 480 nm liposomes. The decrease of x-ray contrast after intratumoral application resulted in a half-life of t50 = 0.05 d for metrizamide solution and t50 = 0.42 d for small liposomes. Large liposomes showed a linear decrease in contrast, the half-life being t50 = 15 d. While small liposomes rapidly leave the tumor, large liposomes rest intact in the tumor for about 30 d. Therefore they fulfill a fundamental prerequisite for intratumoral depots of cytostatics with controlled release.

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The non-ionic X-ray contrast media metrizamide, iopamidol, iohexol, and iopromide do not bind calcium and are less hyperosmolar than the conventional ionic contrast media, for instance amidotrizoate (diatrizoate), iothalamate, or ioglicate. Hence the use of non-ionic contrast media is associated with less undesirable side-effects that are attributable to hypertonicity such as an increase in circulating plasma volume, decreased deformability of red blood cells, damage of vascular endothelium with consequent activation of blood coagulation, the complement system and fibrinolysis, increased release of bradykinin and histamine, cardiac arrhythmias, diuresis, vasodilation and decreased blood pressure, pain and heat sensation. Because of less dilution the quality of imaging is also better. According to the intravenous LD50 in experimental animals the acute toxicity of non-ionic contrast media is lower than that of ionic media. With respect to contrast quality and the rate of side-effects the various non-ionic contrast media appear to be equivalent. Despite their higher price and higher viscosity it is probable that the non-ionic contrast media will replace the classical ionic media, especially in angio- and myelography.

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The penetration into rabbit spinal cord of two nonionic contrast media, iohexol and metrizamide, and a reference tracer, technetium DTPA, were compared. The spinal subarachnoid space was perfused for 4 hours with a CSF solution to which technetium DTPA and either iohexol or metrizamide had been added. The contrast media and technetium DTPA concentrations reached a plateau level in CSF outflow within 80 minutes. The contrast media concentrations in CSF were higher than the technetium DTPA (P less than .001). In the cord tissue, technetium DTPA reached higher concentrations than the contrast media (P less than .001), and iohexol reached higher concentrations relative to technetium DTPA than metrizamide (P less than .001). The mean contrast media distribution volumes in the thoracic cord were 13% (iohexol) and 12% (metrizamide). The smaller distribution volume observed for metrizamide could be related to the larger effective size of "associated" metrizamide molecules or an interference with diffusion perhaps related to binding to glucose carriers.

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Six female adult Macaca mulatta monkeys were made dependent upon morphine sulfate and were implanted with a chronic indwelling needle in the lateral ventricle of the brain for sterile intraventricular injections. Both beta-endorphin and morphine, in a dose dependent manner given intraventricularly suppressed the signs of 14 hour acute morphine abstinence. On a molar basis, beta-endorphin was more active than morphine in suppressing the signs of morphine abstinence. When given intravenously in much larger doses, beta-endorphin was ineffective in contrast to morphine which was effective in suppressing abstinence.

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The diffusion of water-soluble contrast media (CM) into the extracellular space of the central nervous system following injection into the subarachnoid space has previously been shown. As a result of this, water-soluble CM will come in direct contact with the neurons and may interfere with their normal function. The toxic effects would thus be a result both of the molecular properties of the CM as well as the local tissue concentration. The neuronal tissue uptake and clearance of metrizamide in rabbits following lumbar myelography was described in a previous study by our group. This study indicated some retention of metrizamide in the spinal cord probably as a result of binding of the CM to the cell membrane. The mechanism for this has not yet been shown although it may relate to the binding of metrizamide via its 2-deoxy-D-glucose (2-DG) portion and the specific glucose membrane carrier. The present investigation was performed to evaluate the diffusion kinetics of a new non-ionic CM. With iohexol, which lacks a 2-DG component in its molecule a direct relationship between the neural tissue and CSF concentration was found which seems to follow a simple diffusion model. Since iohexol shows no sign of entrapment in the tissue, the contact time for neurons will be shorter than that seen with metrizamide assuming that their rate of drainage from the CSF is identical.

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We report two patients in whom brain penetrance of grey matter by metrizamide, introduced for myelography thirty hours earlier, mimics the periventricular lucency of white matter disease on cranial computed tomography.

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The concentrating mechanisms affecting contrast medium in the distal nephron have been studied using three contrast media of differing osmolality (monomer, dimer and non-ionic compounds) in antidiuretic dogs with three different basal solute excretion rates. Evidence has been obtained that, under conditions of contrast medium osmotic diuresis, the loop of Henle generates more free water. If the free water generated in response to the contrast medium solute load is reabsorbed in the collecting ducts, this can in part mask the different proximal tubular effects of contrast media of different osmolality. However, as basal solute excretion increases, the compensatory mechanism is overwhelmed and urine osmolality falls exponentially. At the higher basal solute excretion rates, different osmolar loads of contrast medium no longer produce different urine osmolalities and different urine contrast medium concentrations. It is therefore considered unlikely that contrast media of reduced osmolality will produce significant increases in urine contrast medium concentration under the conditions of solute diuresis which occur in renal failure.

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The excretory behavior of nine nephrotropic contrast agents with varying physicochemical properties such as charge, lipophilicity, and molecular size was investigated. Renal clearance in comparison with inulin was determined by means of the continuous infusion method. Each contrast agent was infused at three dose levels in four to six rabbits. The investigations show that tubular transportation in proportion to glomerular filtration decreases with increasing dosages of all the contrast agents. Thus, with the highest concentration in plasma all contrast agents are eliminated at more or less the glomerular filtration rate (GFR). After administration of the low dosages the following differences are found: 1) Net tubular secretion increases for the monomeric contrast agent acids with increasing lipophilicity, in the order diatrizoate congruent to iothalamate less than iodamide less than acetrizoate. 2) The clearance studies do not reveal any tubular secretion or reabsorption for a hydrophilic cationic contrast agent. 3) The nonionic contrast agents do not show net secretion. The more lipophilic they are, the more they are reabsorbed. 4) Two dimeric contrast agents also do not reveal any tubular secretion. They seem to be reabsorbed more than monomers with the same charge.

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Forty anaesthetized rabbits were injected subarachnoidally with equimolal doses, 0.48 mmol/kg body weight (= 185 mg I/kg), of iohexol and metrizamide, administered in solutions iso- and hyperosmolal to the CSF. Concentrations of Na, K, Ca and Mg in the CSF were determined before and 3 hours after the contrast medium injections. Iso-osmolal iohexol caused a uniform decrease in all four cations, which was believed to be a simple dilution. The hyperosmolal solution of iohexol caused a significant relative increase in Ca compared with Na, K and Mg. Both metrizamide solutions caused an increase in Ca over its pre-injection level and a significant relative increase of both Ca and Mg as compared with Na and K. The changes caused by the three latter solutions were consistent with an increase in the blood-brain barrier permeability.

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Neurotoxicity from subarachnoid contrast media is probably related to their specific pharmacologic effects and to their penetration into the central nervous system. The lack of a barrier between the cerebrospinal fluid and the extracellular fluid of the brain and cord allows water-soluble contrast media to diffuse into the neural tissue. The aim of this investigation was to develop a method that allows one to quantify the neural tissue penetration for a given contrast medium in relation to the cerebrospinal fluid concentration and contact time and apply this to the use of metrizamide. The result shows a good correlation between iodine concentration in the lumbar cerebrospinal fluid and that in the lumbar cord suggestive of a simple diffusion. When time of sacrifice (contact time) is added as a covariant there is also some indication of retention of metrizamide in the neural tissue. The investigation also demonstrates that the resorption of the contrast medium in the rabbit in this experimental model is mainly in the lumbo-sacral region.

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Responses to intrathecal metrizamide in dogs were found to be age-related: Adult dogs had seizures; 7-week-old dogs appeared stuporous; 6-day-old dogs were clinically unaffected. The brain metrizamide concentrations 4, 6, and 20 hr after intrathecal injection correlated directly with the occurrence and severity of neurotoxic symptoms. Age-related differences in brain metrizamide concentration may be explained by two factors. The first is the failure of current clinical guidelines to adjust the recommended dosage of metrizamide to reflect differences among age groups in brain weight rather than body weight. This error resulted in lower doses/gram of brain weight being given to the puppies. However, the large differences in brain metrizamide concentrations among the three groups of dogs could not be explained solely by differences in the dose. A second factor, physiologic age-related differences in brain penetration, is believed to be operative. The precise nature of these differences is unclear.

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The contrast enhancement of six contrast media (CM) was compared in 13 tissues of the rat after rapid intravenous bolus injection. The rats were sacrificed at 0 and 40 seconds and 2, 5, and 15 minutes after contrast injection. 125I labeled diatrizoate, metrizamide, ioxaglate, iohexol, iopamidol, and a nonionic dimer, iodecol, were each injected at a dose of 612 mg iodine per kg body weight, and iodine concentration (IC) and contrast enhancement were calculated from radioactivity measurements. Higher blood IC values were obtained with the nonionic CM; similar enhancement patterns were seen in the spleen, heart, lungs, and brain. Renal IC was directly related to the number of iodine atoms per ion or molecule of CM. In consequence, renal IC was inversely related to the CM osmolality, but no such correlation was seen with the blood IC. Metrizamide produced the greatest IC in the organs of the gastrointestinal tract. There was no apparent correlation of IC with molecular structure of physicochemical parameters of the CM in any of the other tissues studied.

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Experiments have been made to determine the main route by which radio-iodinated albumin reaches deep cervical lymph from cerebrospinal fluid (c.s.f.) in the anaesthetized rabbit. Other factors, influencing drainage through this pathway, have been investigated. After single injection of [125I]albumin into a lateral ventricle of control rabbits, a mean of 14.8% of the radioactivity lost from brain-c.s.f. was recovered during 6 hr in the lymph of the cannulated jugular trunk of one side. Injection of kaolin into the olfactory fossa or sealing of the cribriform plate with cyanoacrylate glue reduced the recovery of [125I]albumin to 3.3% and 1.9% respectively at 2-3 weeks after the procedure designed to block the cribriform plate. This confirms the traditional view that the major connexions between c.s.f. and deep cervical lymph is via prolongations of subarachnoid space around the olfactory nerves, leading into the interstitial spaces of the nasal submucosa. The dense lymphatic plexus in this tissue is known to drain into the retropharyngeal (deep cervical) lymph nodes. Constant infusion of artificial c.s.f. into a lateral ventricle at 10 microliters/min or 30 microliters/min, in order to approximately double or quadruple flow through the system respectively, decreased the recovery of intraventricular [125I]albumin to 8.1% and 6.9% respectively. It also appeared that the increased c.s.f. pressures induced forced relatively more radioactivity from inside the skull into the c.s.f. spaces of the spinal cord. Maintaining the rabbit prone but at 20 degrees from the horizontal caused recoveries of [125I]albumin in lymph of 17.6% (head-up position and 6.6% (head-down). The amounts of radioactivity in nose and spinal cord markedly increased and decreased respectively in the head-down position. They changed in the opposite directions in the head-up position. The amounts of [51Cr]EDTA, [125I]metrizamide and [14C]inulin in deep cervical lymph were negligible after intraventricular injection. Estimations of the ratio of [51Cr]EDTA/[125I]albumin in various tissues on the pathway into lymph, together with measurements of arterio-venous fluxes across the retropharyngeal nodes, indicate that [51Cr]EDTA passed from c.s.f./lymph into blood within both the nose and the lymph nodes.

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Four new myelographic agents, metrizamide, iopamidol, iohexol, and iotrol, were studied in the subarachnoid space of cynomolgus monkeys. Plain films and computed tomographic scans documented the transport of each material throughout the space and into the brain. At the concentration used (300 mg I/ml), all gave good radiopacity for myelography and delineation of the cerebral subarachnoid space. All four cleared similarly from the ventricular system. Metrizamide, however, penetrated the brain in greater degree and persisted longer than the other three agents. Next in persistence was iopamidol and least, and both statistically equal, iotrol and iohexol.

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Iopromide (Schering, Berlin) is a new nonionic, monomeric contrast medium containing three different substituents on the triiodinated benzene ring. Iopromide exhibits low osmolality and viscosity in aqueous solutions of high concentrations. It has been shown to have a remarkably low intravenous toxicity in mice and rats. Neural tolerance was found to be equal to or better than that of metrizamide when injected in rats intracisternally and intracerebrally, respectively. The effects of iopromide after selective peripheral and cerebral arterial injections in rats were demonstrated to be very moderate at high dosages. The interaction of iopromide with proteins and membranes was found to be considerably low due to its hydrophilicity. Excretion of iopromide is fast and predominantly by the renal route. On the basis of the preclinical profile iopromide is a very promising contrast agent, being most suitable for all angiographic indications, including digital subtraction angiography, urography, and computed tomography.

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Two absorptive pathways for contrast media injected into the lumbar subarachnoid space have been postulated: (1) through the intracranial parasagittal arachnoid granulations and (2) direct absorption through the spinal arachnoid villi into the blood. To study the capacity of the spinal absorptive pathway, serial measurements of metrizamide concentrations in blood serum and urine were obtained before and after lumbar intrathecal injection of contrast medium in four patients with arrested intracranial blood circulation ("brain death") and intracranial pressure exceeding systolic blood pressure who had no circulation of cerebrospinal fluid from the spinal subarachnoid space to the parasagittal arachnoid granulations. These measurements indicated a high capacity of the spinal absorptive pathway for metrizamide elimination.

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