METABOLISM OF γ-HYDROXY-[1-14C] BUTYRATE BY RAT BRAIN: RELATIONSHIP TO THE KREBS CYCLE AND METABOLIC COMPARTMENTATION OF AMINO ACIDS

Abstract— Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ-hydroxy-[1-¹⁴C]butyric acid (GHB) or [1-¹⁴C] succinate. The loss of isotope (as ¹⁴CO₂) was similar for both substances. The [1-¹⁴C]GHB metabolites lost 75% of the label and the [1-¹⁴C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1-¹⁴C]GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the a-amino group. These results also indicate that the label from [1-¹⁴C]GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radiolabelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments preferentially metabolize GHB. It was found that for [1-¹⁴C]GHB this ratio was 4.20 ± 0.18 (S.E. for n = 7) and for [l-¹⁴C]succinate this ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

CONVERSION OF [1-14C]PALMITIC ACID TO [1-14C]HEXADECANOL BY DEVELOPING RAT BRAIN CELL-FREE PREPARATIONS

—The conversion of [l-¹⁴C]palmitic acid to [1-¹⁴C]hexadecanol has been demonstrated with a cell-free system from developing rat brain. ATP, Coenzyme A and Mg²⁺ were required for the activity. Fatty aldehyde was found to be an intermediate in this reaction. The conversion of fatty acid to fatty alcohol was mainly localized in the microsomal fraction and the formation of hexadecanol showed absolute specificity towards NADPH while fatty aldehyde was formed even in the absence of exogenous reduced pyridine nucleotides. The brain microsomes showed maximal activity with stearic acid and the activities with palmitic and oleic acids were 65% and 38% respectively of that with stearic acid. This enzymic reduction increased with age and showed a maximum in the 15-day old rat brain.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2-14C]MEVALONIC ACID, SODIUM [2-14C]ACETATE AND [U-14C]GLUCOSE INTO 15-DAY-OLD RATS

The distribution of [¹⁴C]-labelled material into subcellular fractions of 15-day-old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2-¹⁴C]sodium acetate, [U-¹⁴C]glucose and [2-¹⁴C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U-¹⁴C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U-¹⁴C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U-¹⁴C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2-¹⁴C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2-¹⁴C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.     

THE EFFECTS OF CARBAMYLCHOLINE ON INCORPORATION IN VIVO OF [1-14C]ARACHIDONIC ACID INTO GLYCEROLIPIDS OF MOUSE BRAIN

Abstract— The effects of carbamylcholine on incorporation of [1-¹⁴C]arachidonate into the glycerolipids in mouse brain synaptosome-rich and microsomal fractions were examined at 1, 3 and 10 min after intracerebral injection of the labeled precursor. When carbamylcholine was included with the labeled arachidonate, there was a decrease in the proportion of labeled fatty acid incorporated into the phospholipids. Among the phospholipids in the synaptosome-rich fraction, a decrease in incorporation of radioactivity into diacyl-glycerophosphoinositols and diacyl-glycerophosphocholines was observed at 1 and 3 min after injection. A decrease in labeling of diacyl-glycerophosphoethanolamines and diacyl-glycerophosphocholines in the microsomal fraction was observed at 3 and 10 min after injection. The decrease in phospholipid labeling was marked by an increase in labeling of diacylglycerols which was observed initially in the synaptosome-rich fraction, but also in the microsomal fraction at later time periods. Other lipid changes included an increase in triacylglycerol labeling which was found in the synaptosome-rich fraction and an increase in phosphatidic acid labeling which was found in the microsomal fraction. Results of the in vivo study have demonstrated changes in brain lipid metabolism during carbamylcholine stimulation. Furthermore, these changes appear to be initiated mainly in the synaptosome-rich fraction.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

THE INCORPORATION OF [1-14C]LINOLENATE INTO LIPIDS OF DEVELOPING RAT BRAIN DURING ESSENTIAL FATTY ACID DEPRIVATION

Abstract— Twenty-one-day old essential fatty acid (EFA) deprived rats incorporated about twice the radioactivity from [1-¹⁴C]linolenate into brain lipid fractions as did controls. At 5 min after injection, 2/3 of the radioactivity was associated with the less polar lipid fraction of both control and EFA deprived animals. By 30 min after injection, 70% of the radioactivity was in the phospholipid fraction. This value increased to 90% at later time points.
The specific activity of brain phospholipids from EFA deprived rats was always greater than that of controls. This held true for the individual phosphatide fractions also. In general, phosphatidylcholine (PC) was labeled most rapidly. With increasing time intervals, radioactivity was transferred to phospha-tidylethanolamine (PE) and phosphatidylserine + phosphatidylinositol (PS + PI).
The transfer of fatty acid radioactivity into phospholipid and the distribution of radioactivity among individual phosphatides did not appear to be affected by the dietary state. However, the total amount of radioactivity incorporated was related to the amount initially retained by brain after injection. Our data suggest that one or more components of the less polar lipid fraction may act as a 'trap' or reservoir for fatty acids which are required for phospholipid synthesis.     

METABOLISM OF MALONIC ACID IN RAT BRAIN AFTER INTRACEREBRAL INJECTION

Labeled malonic acid ([1-¹⁴C] and [2-¹⁴C]) was injected into the left cerebral hemisphere of anesthetized adult rats in order to determine the metabolic fate of this dicarboxylic acid in central nervous tissue. The animals were allowed to survive for 2, 5, 10. 15 or 30min. Blood was sampled from the torcular during the experimental period and labeled metabolites were extracted from the brain after intracardiac perfusion. There was a very rapid efflux of unreacted malonate in the cerebral venous blood. Labeled CO₂ was recovered from the venous blood and the respired air after the injection of [1-¹⁴C]malonate but not after [2-¹⁴C]malonate. The tissue extracts prepared from the brain showed only minimal labeling of fatty acids and sterols. Much higher radioactivity was present in glutamate, glutamine, aspartate, and GABA. The relative specific activities (RSA) of glutamine never rose above 1.00. Aspartate was labeled very rapidly and revealed evidence of ¹⁴CO₂ fixation in addition to labeling through the Krebs cycle. GABA revealed higher RSA after [1-¹⁴C]malonate than after [2-¹⁴C]malonate. Sequential degradations of glutamate and aspartate proved that labeling of these amino acids occurred from [1-¹⁴C] acetyl-CoA and [2-¹⁴C] acetyl-CoA, respectively, via the Krebs cycle. Malonate activation and malonyl-CoA decarboxylation in vivo were similar to experiments with isolated mitochondria. However, labeled malonate was not incorporated into the amino acids of free mitochondria. The results were compared to data obtained after intracerebral injection of [1-¹⁴C]acetate and [2-¹⁴C]acetate.     

THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U-14C]GLUCOSE AND [U-14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN

Abstract— The incorporation of ¹⁴C into amino acids of the brain was determined at different times after injection of [U-¹⁴C]glucose and [U-¹⁴C]ribose to rats maintained on thiamine-supplemented and thiamine-deficient diets for 22 days.
The ¹⁴C-content of amino acids in the brain of thiamine-deficient rats decreased at times 2–10 min after injection of [U-¹⁴C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U-¹⁴C]ribose.
The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a-oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.
Thiamine deficiency in rats showed a 5% decrease in glutamate ( P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine ( P < 0–01) content of the brain.     

ALTERATION OF TRICARBOXYLIC ACID CYCLE METABOLISM IN RAT BRAIN SLICES BY HALOTHANE

Abstract—

• 1 Metabolism of [2-¹⁴C]pyruvate, [1-¹⁴C]acetate and [5-¹⁴C]citrate in the rat cerebral cortex slices was studied in the presence of halothane. Metabolites assayed include acetylcholine (ACh), citrate, glutamate, glutamine, γ-aminobutyrate (GABA) and aspartate. The trichloroacetic acid soluble extract, the trichloroacetic acid insoluble precipitate and its lipid extract were also studied.
• 2 In control experiments, pyruvate preferentially labelled ACh, citrate, glutamate, GABA and aspartate. Acetate labeled ACh, but to a lesser extent than pyruvate. Acetate also labeled lipids and glutamine. Citrate labeled lipids but not ACh and served as a preferential precursor for glutamine. These data support a three-compartment model for cerebral tricarboxylic acid cycle metabolism.
• 3 Halothane caused increases in GABA and aspartate contents and a decrease in ACh content. It has no effect on the contents of citrate, glutamate and glutamine.
• 4 Halothane preferentially inhibited the metabolic transfer of radioactivity from pyruvate into almost all metabolites, an effect probably not related to pyruvate permeability. This is interpreted as halothane depression of the‘large metabolic compartment’ which includes the nerve endings.
• 5 Halothane increased the metabolic transfer of radioactivity from acetate into lipids but did not alter such a transfer into the trichloracetic acid extract.
• 6 Halothane increased the metabolic transfer of radioactivity from citrate into the trichloroacetic acid precipitate, lipids and especially glutamine. Transfer of citrate radioactivity into GABA was somewhat decreased.
• 7 The differential effects of halothane on acetate and citrate utilization suggest that the ‘small metabolic compartment’ should be subdivided. Therefore, at least three metabolic compartments are demonstrated.
• 8 Halothane did not interfere with the dicarboxylic acid portion of the tricarboxylic acid cycle.

     

IN VIVO STUDIES ON THE METABOLISM OF [15,16-3H]TETRACOSANOIC ACID (LIGNOCERIC ACID) IN ADULT RAT BRAIN

—Adult rats were killed 16 h, 48 h, 6 days and 21 days after intracerebral application of n-[15,16-³H]tetracosanoic acid (lignoceric acid). After incorporation into complex lipids with a strong preference for the ester-bound fatty acids of glycerophospholipids, radioactivity decreased with time. The incorporated activity into the amide-bound fatty acids of sphingolipids was also shown to decrease, with exception of the cerebroside of the hydroxy fatty acid type (cerebron fraction). Only negligible amounts of labelled triglyceride and cholesterol ester could be detected. The fatty acids derived from the complex lipids were analysed by radio gas chromatography. It was revealed that some of the applied labelled lignoceric acid was hydroxylated and incorporated into the cerebron fraction while the rest had their chains shortened. In the latter case all even and odd numbered chain lengths down to C₁₈ and C₁₆ (stearic and palmitic acid) were detected. At this stage, the pool of the degradation products of lignoceric acid is stabilized by the preferred incorporation of fatty acids of these chain lengths into glycerophospholipids. A time-dependent desaturation to oleic acid from stearic acid was observed.     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

METABOLISM OF [14C]LEUCINE AND [14C]ACETATE IN SENSORIMOTOR CORTEX, THALAMUS, CAUDATE NUCLEUS AND CEREBELLUM OF THE CAT

Abstract— —In the head of the caudate nucleus, the relative specific activity of glutamine (glutamic acid specific activity = 1) was less than 1 with intravenous [¹⁴C]leucine as the tracer metabolite. This is in contrast to observations made in other brain areas (cortex, hippocampus, thalamus, pons, and medulla) where the relative specific activity of glutamine was greater than 1. This is also in contrast to findings when [l-¹⁴C]acetate was utilized as the tracer; under these conditions, in all brain areas, including the head of the caudate nucleus, the relative specific activity of glutamine was greater than 1. It is inferred that the differences in metabolism of [¹⁴C]leucine and [¹⁴C]acetate in the head of the caudate from that in other brain areas reflect differences in compartmentation of the glutamate-glutamine system.     

UPTAKE AND RELEASE OF EXOGENOUS [1-14C]ACETYLCHOLINE BY BRAIN CORTEX SLICES FROM THE RAT

Abstract— Radioactive acetylcholine ([¹⁴C]ACh) that is taken up by rat cerebral cortex slices, incubated aerobically in a physiological saline-glucose paraoxon-[¹⁴C]ACh medium, apparently by a passive diffusion process at concentrations > 1 mm consists essentially of two forms, a readily exchangeable and releaseable or mobile form, and a bound or retained form, poorly (or not) exchangeable. The quantity of retained ACh consists of a considerable fraction of that taken up amounting to 54% with external 0.1 mm -[¹⁴C]ACh and about constant, 27%, for the range 5-50mm -[¹⁴C]ACh. All its ACh is released on homogenization with 0.1 n -perchloric acid or on tissue disintegration in distilled water. The cerebral uptake of ACh differs basically from that of urea as there is no retention of the latter following its uptake. Cerebral cortex slices are superior to those of cerebellar cortex, subcortical white matter, kidney cortex, liver and spleen in taking up and retaining [¹⁴C]ACh. Deprivation in the incubation media of glucose or Na⁺ or Ca²⁺. or the presence of dinitrophenol, whilst causing little change in ACh uptake, induces considerable changes in swelling and ACh retention; the greater the amount of swelling the smaller is that of retention. It seems that the latter is segregated in compartments characterized by a low permeability to exogenous ACh. About half of it is independent of changes in incubation conditions whilst the other half enters the compartment by an Na⁺, Ca²⁺ and energy-dependent process. At least part of the retention is neuronal as it is diminished by protovera-trine, the diminution being blocked by tetrodotoxin. Mobile ACh (i.e. total uptake minus retained ACh) is largely unaffected by protoveratrine, ouabain, etc. It seems that the retained ACh is directly proportional to the amount of mobile ACh minus the amount that enters with swelling. If the latter is largely glial in location, then the retained ACh is simply proportional to the mobile neuronal ACh. Suggestions are made as to the location of the retained ACh in the brain cells and to the processes involved in its segregation there. Release of retained ACh occurs on change of the Na⁺ gradient. Atropine and d-tubocurarine also diminish the amount of retained ACh but the percentage diminution falls with increase of the concentration of exogenous ACh.     

COMPARISON OF CEREBRAL REGIONAL METABOLISM OF [14C]LEUCINE FOLLOWING THIRD VENTRICLE AND INTRAVENOUS ADMINISTRATION IN THE CAT

Abstract— In the cat, intraventricularly injected [¹⁴c]leucine does not appear to penetrate into the cerebral tissue, whereas intravenously injected [¹⁴c]leucine readily penetrates the blood-brain barrier. The latter route of administration of [¹⁴c]leucine produces rather uniform distribution of radioactivity in cortical and subcortical regions as well as diencephalic, lower brain stem, and cerebellar regions. Data consistent with compartmentation of the glutamate-glutamine system were observed in all regions except the cerebellum and head of the caudate nucleus. In the latter two areas, the ratios of the specific activity of glutamine to glutamic acid was less than 1, whereas in all other areas it was greater than 1. The turnover rate of the brain protein was fastest in the cerebellum and neocortex and slowest in the caudate nucleus and in the pons and medulla.     

METABOLISM OF d-[U-14C]RIBOSE IN RAT TISSUES

Abstract— d -[U-¹⁴C]Ribose injected subcutaneously into the rat enters the blood, liver and brain. At 30 min after injection 40-70 per cent of the radioactivity in the brain was found in amino acids and only 2-6 per cent in free sugars. In contrast, free sugars (mainly glucose) and carboxylic acids accounted for most of the radioactivity in liver and blood. Evidence for the entry of [U-¹⁴C]ribose into the brain was obtained by intracarotid or intravenous injection of [U-¹⁴C]ribose after interrupting the blood supply to the liver and kidney. Under these conditions the radioactivity in the brain was found in amino acids, carboxylic acids and ribose; no significant amount of [¹⁴C]glucose was detected in brain or heart. It is concluded that ribose is metabolized directly in vivo in the brain. d -[U-¹⁴C]Ribose was metabolized also by brain slices in vitro to form ¹⁴C-labelled amino acids and carboxylic acids; the rate was equivalent to the utilization of 0.65 μ mol of ribose/g/h. The specific radioactivity of glutamine and of γ -aminobutyrate was similar to or higher than that of glutamate in the brain. These results are discussed in the context of metabolic compartments.     

SIMULTANEOUS INCORPORATION OF ORALLY ADMINISTERED [9, 10-3H2]OLEIC AND [1-14C]LINOLEIC ACIDS INTO LIPIDS OF THE ADULT RAT BRAIN

—The incorporation of an orally administered mixture of [9,10-³H₂joleic acid and [1-¹⁴C]linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin-cholesterol acyl transferase. In neural tissue, a considerable amount of [1-¹⁴C]linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.