BINDING OF [3H]KAINIC ACID, AN ANALOGUE OF l-GLUTAMATE, TO BRAIN MEMBRANES

—The specific binding of [³H]kainic acid to synaptic membranes from rat brain was saturable with a dissociation constant of about 60 nm . The apparent maximal number of binding sites was about 1 pmol/mg protein. The most effective displacer of specific [³H]kainic acid binding was quisqualic acid, a powerful excitant which is structurally similar to l -glutamate. However, quisqualic acid was one-third as potent a displacer as kainic acid itself. l -Glutamate was the next potent in displacing [³H]kainic acid binding, but also was less effective (1/25) than kainic acid itself. All other compounds including suspected neurotransmitters were at least an order of magnitude lower in potency compared to l -glutamate. When various tissues and brain regions were tested for specific [³H]kainic acid binding, we found the specified binding was localized to grey matter in the brain. In studies of subcellular fractionation of the brain, we found that crude synaptosomal membrane preparations were most enriched in specific [³H]kainic acid binding. Specific [³H]kainic acid binding in various regions of the rat brain varied 5- to 6-fold.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

Transport and distribution of homocarnosine after intracerebroventricular and intravenous injection in the rat

Rats were injected intracerebroventricularly (i.c.v.) or i.v. with [¹⁴C]homocarnosine (250 nmol). Distribution of the dipeptide in brain structures, transport from the brain to the blood, distribution in peripheral organs, and excretion in the urine were studied by measuring radioactivity in tissue, plasma, and urine samples by liquid scintillation counting 15–120 min after injection. After i.c.v. injection, [¹⁴C]homocarnosine was taken up into all parts of the brain investigated (highest uptake in structures close to the site of injection), it was transported to the blood, and radioactive substances were found in low concentration in muscle, spleen, and liver, in high concentration in the kidneys, and very high concentration in the urine. Investigations using high pressure liquid chromatography (HPLC) showed that no degradation took place in the brain, all radioactivity was found in the homocarnosine fraction. In the plasma 86% of the radioactivity was found in the GABA fraction presumed to be formed by cleavage of the peptide, while in the kidneys 35% and in the urine 40% was found in the GABA fraction. After i.v. injection of [¹⁴C]homocarnosine, no radioactivity was measured in hippocampus, striatum, cerebellum and cerebral cortex 15 min after injection, however, 60 min after injection a very low activity was detected in these structures (estimated intravascular radioactivity subtracted). A low activity was also measured in the spinal cord both 15 and 60 min after injection. When homocarnosine and GABA were separated on HPLC, all radioactivity in brain tissue was found in the GABA fraction, indicating either that [¹⁴C]homocarnosine did not cross the blood-brain barrier in amounts that could be measured with the method used, or that peptide entering the brain was rapidly transported back to the blood. [¹⁴C]Homocarnosine was not taken up either into crude synaptosomal preparations from hippocampus, striatum, cerebellum, cortex and spinal cord, or into slices prepared from the hippocampus and striatum. Transport from the brain to the kidneys and excretion in the urine seems to be a major route for disposal of this peptide in the rat.     

Accumulation,elimination, release and metabolism of pipecolic acid in the mouse brain following intraventricular injection

Following i.c.v. (intracerebral ventricular) injections ofd,l-[³H]pipecolic acid (PA), it is reabsorbed from the ventricles and redistributed to various brain regions. The highest accumulation is found in three brain regions ipsilateral to the injection site, hippocampus, neocortex, striatum, and in the diencephalon. Following preloading in vivo, the radioactivity is released from hippocampus slices in the perfusion medium after depolarization induced by high K⁺. During perfusion with a Ca⁺⁺ free medium containing EGTA, a significant reduction of release is observed.The radioactivity ofd,l-[³H]PA in the brain shows a more rapid phase of decrease from 0 to 2 hours and a slower phase from 2 to 5 hours. At 5 hours, only 28% radioactivity, represented mainly by PA, is left in the brain. Kidney secretion represents the major route of elimination of the injected PA. The presence of -aminoadipic acid both in brain and urine was observed. Probenecid (200 mg/kg) significantly increases the accumulation of i.c.v. injectedd,l-[³H]PA in brain and kidney. The presence of a regional accumulation of PA in certain brain regions, its metabolism in brain, its enhanced retention following probenecid administration and its Ca⁺⁺ dependent release following high K⁺ stimulation, all constitute indirect evidence for a neuronal localization of this brain endogenous iminoacid.     

Accumulation and metabolism of pipecolic acid in the brain and other organs of the mouse

The long-term accumulation of pipecolic acid, as well as its disappearance following exogenous administration was studied in brain and other organs of the mouse. Mice were pulse-injected intraperitoneally or intravenously with 1Ci[³H]D,l-pipecolic acid (6.9 nmol/mouse=2.9 g/kg). The total radioactivity retained in tissues was measured in brain, liver, and kidney, as well as in plasma during the period 1 min to 24 hr. TLC separation of DNP-derivatives was performed. Three features of the pattern of retention of pipecolic acid are most salient; first the rapid accumulation in brain, second the rapid secretion of this compound in the urine, and third the long-lasting steady levels of radioactivity maintained in brain.Sixty minutes after i.v. injection, the brain/plasma ratio is approximately 0.2 and approaches unity only at 5 hr. Following intraperitoneal injection the percent recovered as pipecolic acid in brain is 78% at 30 min and 71% at 120 min, suggesting a slow metabolic activity. Liver shows a different trend than brain with a slower accumulation and a faster disappearance. Kidney shows a pattern similar to plasma with a rapid secretion of radioactivity into urine which correlates well with the exponential decrease in plasma and urine. The administration of probenecid significantly increases radioactivity due to pipecolic acid in brain, liver, and urine. Formation of -aminoadipic acid, a known metabolite of pipecolic acid, can be demonstrated in kidney 30 min after intraperitoneal injection. The present data together with results obtained previously with intracarotid injections suggest that pipecolic acid is taken up in the mouse brain from the circulation. Most of the pipecolic acid taken up is rapidly removed through the circulation and secreted in the urine; however, a small part is retained and probably metabolized by brain and kidney.     

Regional distribution of [3H] kainic acid after intraventricular injection

[³H] Kainic acid was administered intraventricularly to rats at a dose that selectively destroys the pyramidal cells of hippocampal area CA3. Only about one-third of the injected radioactivity was recovered in the brain 15 min later, but the residual radioactivity disappeared at a much slower rate. [³H]-Kainic acid distributed rather evenly throughout the brain; there was no correlation between accumulation of radioactivity and neurotoxicity. Almost 90% of the radioactivity in sucrose homogenates was recovered in the high-speed supernatant. No cerebral metabolism of [³H] kainic acid was detected by thin-layer chromatography. These data rule out the possibility that a lethal accumulation of the toxin by hippocampus accounts for the preferential vulnerability of hippocampal pyramidal cells.     

Diffusion of intracerebrally injected [1-14C]arachidonic acid and [2-3H]Glycerol in the mouse brain

[2-³H]Glycerol and [1-¹⁴C]arachidonic acid were injected into the region of the frontal horn of the left ventricle of mice and were distributed rapidly throughout the brain. After 10 sec, most of the radioactive fatty acid was found in the hemisphere near the injection site; after 10 min, it was recovered in similar proportions in the cerebellum and brain stem. [2-³H]Glycerol showed a heterogeneous distribution, with most of the label remaining in the left hemisphere even after 10 min. On a fresh weight basis, cerebrum, cerebellum, and brain stem were found to contain similar amounts of labeled glycerol. However, the amount of [1-¹⁴C]arachidonate in cerebrum was only 50% of that recovered from cerebellum or brain stem. Brain ischemia or a single electroconvulsive shock reduced the spread of the label, producing an accumulation of radioactivity in the injected hemisphere, except for an increase in [2-³H]glycerol in the brain stem during ischemia. Despite the significant decrease in available precursor in the cerebellum and brain stem after electroshock, the amount of label incorporated into lipids was not altered in these areas and only slightly diminished in the cerebrum.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Evidence for pituitary-brain transport of a behaviorally potent ACTH analog.

Following intrapituitary injection of ³H-ACTH 4–9 analog, the radioactivity of various brain regions was determined in intact rats and in rats with the pituitary stalk cut one or eight days previously. The regional distribution of radioactivity in the brain was also investigated after intravenous and intrasellar administration. Intrasellar and intrapituitary administration resulted in significantly higher radioactivity levels in the brain than did intravenous injection of an equimolar dose of labeled peptide. Intrapituitary injection resulted in an uptake with clear regional differences and which was highest in the hypothalamus. Twenty four hours after stalk section the uptake of radioactivity in the hypothalamus, but not in other brain regions was markedly depressed. Hypothalamic uptake, however, was restored at eight days after stalk section. The results suggest a significant flow of radioactivity from the pituitary to the brain, particularly to the hypothalamus. Transport to the hypothalamus is presumably partly vascular via the stalk. Transport to other brain areas may occur via the cerebrospinal fluid, but a neural route cannot be excluded.     

Intrastriatal kainic acid elicits functional supersensitivity of muscarinic receptors regulating dopamine release

Acetylcholine enhanced in a concentration-dependent way the K⁺ (15 mM)-evoked release of [³H]dopamine from synaptosomes isolated from rat corpus striatum and prelabeled with the radioactive catecholamine. The concentration-effect curve of ACh obtained in presence of 1.2 mM Ca²⁺ was progressively shifted to the left when [Ca²⁺] was lowered to 0.4 and to 0.2 mM. Intrastriatal injections of kainic acid reduced (70%) the uptake of [³H]choline in synaptosomes prepared 8 days after the lesion but did not affect significantly the uptake of [³H]dopamine. Also the release of [³H]dopamine evoked by K⁺ was minimally affected by kainic acid treatment. In contrast, acetylcholine (tested in presence of 1.2 or 0.2 mM Ca²⁺) was much more effective in enhancing [³H]dopamine release in synaptosomes from kainic acid-lesioned than from unlesioned striata. The results suggest that muscarinic receptors located on dopamine nerve terminals undergo supersensitivity following intrastriatal kainic acid injection.     

The incorporation of [14C]palmitic acid into the lipids of mouse brain in vivo

Abstract— The half-life of free [¹⁴C]palmitic acid injected intracerebrally into C57BL/10J mice was less than 5 min. The rapid disappearance of radioactivity as palmitic acid was accompanied by increases in the radioactivity of the phosphatidic acids and the diacyl-glycerols. The peak specific radioactivity of the diacylglycerols occurred at about 6-8 min after injection. The triacylglycerols, phosphatidyl ethanolamines and phosphatidyl cholines exhibited increasing amounts of radioactivity during the first 40 min. At 160 min after injection, the distribution of radioactivity was similar to the pattern observed at 12 h. The biosynthetic pathway through the phosphatidic acids and the diacylglycerols to triacylglycerols, phosphatidyl ethanolamines and phosphatidyl cholines is apparently the major pathway in vivo for the esterification of free fatty acids in the brain.     

Are nociceptin(1‐13)NH2 and its structural analogue [ORN9]nociceptin(1‐13)NH2 able to affect brain antioxidant status in control and kainic acid‐treated rats?

In‐vivo effects of nociceptin (N/OFQ(1‐13)NH₂) on the levels of lipid peroxidation and cell enzyme (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non‐enzyme (glutathione) antioxidants in brain of control and kainic acid‐treated rats were studied. N/OFQ(1‐13)NH₂ effects were compared with those of its structural analogue [Orn⁹]N/OFQ(1‐13)NH₂. Kainic acid (25 µg, i.c.v) increased the lipid peroxidation (4 and 24 h after kainic acid treatment) and decreased the glutathione level (1 h after kainic acid injection). We failed to find, any changes in antioxidant enzyme activities, independently of the time of kainic acid treatment. At the background of kainic acid‐effects, N/OFQ(1‐13)NH₂ and [Orn⁹] N/OFQ(1‐13)NH₂, injected 30 min before kainic acid, had no effects on all parameters, tested in brain. In addition, the neuropeptides did not change the antioxidant status in brain of control animals. It might be concluded that N/OFQ(1‐13)NH₂ and [Orn⁹]N/OFQ(1‐13)NH₂ have neither pro‐ nor anti‐oxidant activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Dehydroepiandrosterone sulphate in rat brain: incorporation from blood and metabolism in vivo

The incorporation of [7-³H]dehydroepiandrosterone[³⁵S]sulphate into brain tissue elements from the circulatory system and its metabolic fate in the brain were studied in developing rats. Approximately 0.037 % of [³H] and 0.023% of [³⁵S] were incorporated into the brain within 15 min after the intracardiac injection of the labelled steroid. More than one-half of the incorporated [³H] was recovered as free steroid, whereas the rest was recovered as sulphate. The ³H/³⁵S ratio in the sulphate fraction suggested that the sulphate entered the brain with the sulphate linkage intact. Upon intracerebral injection of the double-labelled steroid, approximately 6 per cent of the radioactivity was recovered in the brain at 30 min after the injection and 1 per cent was recovered at 1 h after the injection. Of the remaining radioactivity recovered from the brain, 5 per cent was found in the free steroid fraction, probably formed by hydrolysis of the sulphate; 90 per cent was in the sulphate ester fraction; and the rest was in the fraction of more polar compounds. To identify the metabolites, [4-¹⁴C]dehydroepiandrosterone sulphate was injected into the rat brain. Significant amounts of radioactivity were found in androstenediol sulphate, which was isolated from the brain. This compound was apparently derived from dehydroepiandrosterone sulphate by reduction of the 17-keto group to a 17β-hydroxyl group without prior hydrolysis. There was suggestive evidence that free androstenediol was also formed in the brain in this experiment.     

Effect of intrahippocampal kainic acid on the behavior of rats and functional state of mitochondria in brain structures

Cognitive processes and functional state of mitochondria in brain structures of Wistar rats were studied after intrahippocampal injection of kainic acid, an agonist of glutamate receptors. A single administration of 0.25 μg kainic acid into the dorsal part of the left and right hippocampi affected task retrieval and decreased inhibition of unrewarded responses. The injection of 0.75 μg kainic acid induced recurrent seizures and completely disorganized animal behavior. The functional state of mitochondria, as an important marker of excitotoxicity, was studied after intrahippocampal injections of kainic acid in the same doses. Kainic acid at 0.25 μg proved to activate the oxidative phosphorylation in hippocampal mitochondria. A higher (epileptogenic) dose of kainic acid inhibited mitochondrial respiration in the frontal cortex, but had an insignificant effect on mitochondrial respiration in the hippocampus. The disturbed interaction between the hippocampal system and frontal cortex after kainic acid administration can be the main factor of the revealed cognitive dysfunctions.     

Synthesis and evaluation of [11C]cyanoimipramine

[¹¹C]Cyanoimipramine has been prepared by methylation of the desmethyl cyanoimipramine with [¹¹C]methyl iodide. The chemically and radiochemically pure labelled product was obtained with a high specific activity (> 300 mCi/μmol). When ¹¹C (or ³H)-cyanoimipramine was intravenously administered in mice, high accumulations were shown in brain and lung. Thirty minutes after injection of the tracer, differences were found in the radioactivity between the cerebral cortex and the cerebellum. The regional distribution of radioactivity in the rat brain 30 min after i.v. injection of [¹¹C]cyanoimipramine was also examined, and the radioactivity was high in receptor rich areas (striatum, cerebral cortex etc.) but low in receptor poor area (cerebellum). The in vivo stability of [³H]cyanoimipramine was quite stable in the mouse brain for at least 30 min. Thirty minutes after injection, the radioactivity in the cerebral cortex of the carrier-added state was reduced as compared with the carrier-free state. Taken together, the in vivo specific binding of [³H]cyanoimipramine in the cerebral cortex was estimated at about 40–50% of the total radioactivity. Furthermore, the distribution of [³H]cyanoimipramine in the mice forced to swim was examined. Significant changes in the distribution of [³H]cyanoimipramine were observed in the cerebral cortex.     

Synthesis of taurine in rat liver and brain in vivo

The in vivo formation of taurine and the analysis of labeled taurine precursors was examined in rat brain and liver at different times after an intracisternal injection of [³⁵S]cysteine and an intraperitoneal injection of [³H]cysteine, simultaneously administered. The distribution pattern of radioactivity was similar in liver and brain. Most of the labeling in both organs (85% in brain and 80% in liver) was recovered in glutathione (oxidized and reduced), cysteic acid, cysteine sulfinic acid, hypotaurine, cystathionine, and a mixed disulfide of cysteine and glutathione. The relative rates of labeling of cysteine sulfinic acid and taurine in liver and brain suggest than in vivo, liver possesses a higher capacity for taurine synthesis than brain. A small amount of [³H]taurine was detected in brain after intraperitoneal injection of [³H]cysteine. The time of appearance of this [³H]taurine as well as the fact that it occurs when [³H]cysteine is not detectable in brain or plasma suggests that it was probably not synthesized in brain from labeled precursors but formed elsewhere and transported into the brain through an exchange process.     

Disposition of fucose in brain

Abstract— Labelled fucose administered to rats in vivo was rapidly incorporated into brain glycoproteins, but not into any other brain constituents, including glycolipids and acid mucopolysaccharides. Maximum incorporation of tritium-labelled fucose into brain glyco-proteins occurred 3–6 h after intraperitoneal injection in young or adult rats, and the half-time for the turnover of glycoprotein-fucose in young rats was approximately 2 weeks. Within 3 h after the administration of either [1-³H]fucose or fucose generally labelled with tritium, 75 per cent of the total acid-soluble radioactivity in plasma and brain was found to be volatile, and by 24 h after injection more than 90 per cent of the acid-soluble radioactivity was volatile. The tritium in labelled fiicose appears to undergo arapid exchange reaction with hydrogen atoms in body water, although the tritium in fucose glycosidically- linked to glycoproteins is biologically stable. The rapid disappearance of labelled free fucose from the plasma and tissues of the rat precludes the possibility of any significant degree of reutilization of labelled precursor, and provides support for other data indicating that the turnover of fucose in brain glycoproteins is relatively slow in comparison to that of hexosamine and sialic acid. Activities of α-L-fucosidase in rat brain, with pH optima at 40 and 6.0, had essentially the same K_m (4 × 10^?4 M and 3.2 × 10^?4 M, respectively) with p-nitrophenyl-α-L-fucopyranoside as substrate. Activities of both were competitively inhibited by L-fucose. However, the K_t measured at pH 4 (1.9 × 10^?2) was almost ten times greater than that measured at pH 6 (1.5 × 10^?4).     

Cystathionine in rat brain: Catabolism in vivo

The content of cystathionine was measured in 35 rat brains; the range was 10–120 nmol/g wet weight and thus the variability of cystathionine content in rat brain was emphasized. The regional distribution of cystathionine was also determined: the highest level was found in cerebellum; the lowest level was observed in the white and gray matter of the hemispheres. These results are different from those obtained in other species. The radioactive metabolites formed froml-[³⁵S]cystathionine injected intracisternally were measured in brains of rats killed at the following times after injection: 0.25, 1, 2, 4, 6, 9, 16, and 27 hr. The radioactivity was found both in the proteins and in the acid-soluble fraction. In the acid-soluble fraction the radioactivity was found in various ninhydrin-reacting compounds: (cysteic + cysteine sulfinic) acid, taurine, reduced and oxidized glutathione, cystine, cystathionine, and a compound tentatively identified as the mixed disulfide of cysteine and glutathione. The radioactivity of cystathionine decreased exponentially between the 1st and the 27th hour after injection and its half-life was estimated to be about 5 hr. The radioactivity in the other ninhydrin-reacting compounds increased until the 9th hour after injection, then decreased. Half of this radioactivity was present in reduced glutathione, the rest being shared equally between: (cysteic + cysteine sulfinic) acid, taurine, and the mixed disulfide. It is worthwhile to note that the radioactivity in the cystine fraction was always very low.     

Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.     

In vivo metabolism of 3-ketoceramide in rat brain

Eight hours after intracerebral injection of a double-labeled 3-ketoceramide⁴, [1-¹⁴C]lignoceroyl 3-keto [1-³H]sphingosine, various brain sphingolipids were isolated. Free ceramide and the ceramide portions of nonhydroxy cerebroside and sphingomyelin were further fractionated into subgroups containing longer-chain or shorter-chain fatty acids. Nonhydroxy ceramide, nonhydroxy cerebroside and sphingomyelin containing longer-chain fatty acids had significant quantities of radioactivity with ³H/¹⁴C ratios similar to each other but lower than that of the injected material. The sphingolipids containing shorter-chain fatty acids were also significantly labeled; however, the ³H/¹⁴C ratios were much higher than that of the injected material. Hydroxy-ceramide and sulfatides contained very little radioactivity. However, hydroxy-cerebroside contained an amount of radioactivity comparable to that of the longer-chain nonhydroxy cerebroside with a similar ³H/¹⁴C ratio. It is proposed that the injected 3-ketoceramide was converted into ceramide, cerebroside, and sphingomyelin and that the fatty acids of these lipids were partly replaced by other fatty acids during the metabolic conversions.