Application of simultaneous determination of 3H, 14C, and 22Na by liquid scintillation counting to the measurement of cellular ion-transport.

A liquid scintillation counting method for simultaneous determination of three radioactive nuclides (3H, 14C, and 22Na) of biological interest was studied. By comparing the beta spectra of the three nuclides, their counting energy ranges, A, B, and C, were determined. 22NA was set high enough to avoid any spillover counts from lower-energy nuclides. Region A for 3H was set to maximize the counting efficiency. A good correlation between the counting efficiency for 22Na in region C and the counting efficiency of other nuclides in all regions was obtained. Prior to 3H and 14C dpm calculations, the 22Na counts spilled down in regions A and B were subtracted from the total counts in regions A and B. A simple linear equation was then used to compute 3H and 14C dpm. Findings show that the method presented is adaptable for highly quenched samples up to quenching indices of tSIE = 100. The method is useful for studying the biological transport coupled to Na+.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Determination of catechol-O-methyltransferase activity in brain tissue by high-performance liquid chromatography with on-line radiochemical detection

A sensitive assay for catechol-O-methyltransferase (COMT) activity by high-performance liquid chromatography with on-line radiochemical detection was described. The method was based on the measurement of 3H-labeled 3-O- and 4-O-methylated products of the substrate, 3,4-dihydroxybenzoic acid, using S-adenosyl-L-[methyl-3H]methionine as the methyl donor, or the measurement of 14C-labeled 3-O- and 4-O-methylated products of the substrate, [7-14C]dopamine. The reaction products were determined from the incubation mixture after removal of protein by injecting an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 40% was 0.45 pmol 3H-labeled product, and 0.04 pmol 14C-labeled product with 61% counting efficiency. The method is suitable for assaying membrane-bound and soluble COMT activities in the brain tissue and for calculation of meta/para product ratios.     

An improved scintillation cocktail of high-solubilizing power

A scintillation cocktail containing 25% Triton X-114 in xylene is considered for a broad range of scintillation counting applications. The cocktail gives good counting efficiencies for ³H (47%) and ¹⁴C (93%). It will accept up to 30% (v/v) aqueous sample. The scintillation fluid is also used effectively with samples which are difficult to solubilize, such as the degradation products from the solubilization of polyacrylamide gels. The cocktail can be formulated for less than $2.00 per gallon.     

The metabolic fate of (2-14C)folic acid and a mixture of (2-14C)- and (3'',5'',9-3h)-folic acid in the rat.

The metabolism of [2-14C]folic acid over 13 days and a mixture of [2-14C]- and [3',5',9-3h]-folic acid in rats over a 6-day period is described. Both 14C and 3H are excreted in urine over the 6-day period, but 3H and 14C are only detectable in faeces for 2 days. A breakdown product of folic acid labelled with 3H only was found in some urine samples, but no metabolite corresponding to the part of the molecule containing 14C was detected. These experiments show that in the whole animal a substantial portion of orally administered folic acid undergoes scission shortly after administration [Blair Biochem. J. (1957) 68, 385-387] and that the retained folates are a shortage form for folate monoglutamates.     

The comparative metabolism of 2,6-dichlorothiobenzamide (prefix) and 2,6-dichlorobenzonitrile in the dog and rat

1. A single oral dose of either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile to rats is almost entirely eliminated in 4 days: 84.8-100.5% of (14)C from [(14)C]Prefix is excreted, 67.3-79.7% in the urine, and 85.8-97.2% of (14)C from 2,6-dichlorobenzo-[(14)C]nitrile is excreted, 72.3-80.7% in the urine. Only 0.37+/-0.03% of the dose of [(14)C]Prefix and 0.25+/-0.03% of the dose of 2,6-dichlorobenzo[(14)C]nitrile are present in the carcass plus viscera after removal of the gut. Rats do not show sex differences in the pattern of elimination of the respective metabolites of the two herbicides. The rates of elimination of (14)C from the two compounds in the 24hr. and 48hr. urines are not significantly different (P >0.05) from one another. 2. After oral administration to dogs, 85.9-106.1% of (14)C from [(14)C]Prefix is excreted, 66.6-80.9% in the urine, and 86.8-92.5% of (14)C from 2,6-dichlorobenzo[(14)C]nitrile is excreted, 60.0-70.1% in the urine. Dogs do not show sex differences in the pattern of eliminating the metabolites of either Prefix or 2,6-dichlorobenzonitrile. 3. Dogs and rats do not show species differences in the patterns of elimination of the two herbicides. 4. Prefix and 2,6-dichlorobenzonitrile are completely metabolized; unchanged Prefix and 2,6-dichlorobenzonitrile are absent from the urine and faeces, and from the carcasses when elimination is complete. In the hydrolysed urine of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, 2,6-dichloro-3-hydroxybenzonitrile accounts for approx. 42% of the (14)C, a further 10-11% is accounted for by 2,6-dichlorobenzamide, 2,6-dichlorobenzoic acid, 2,6-dichloro-3- and -4-hydroxybenzoic acid and 2,6-dichloro-4-hydroxybenzonitrile collectively, and 25-30% by six polar constituents, of which two are sulphur-containing amino acids. 5. In the unhydrolysed urines of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, there are present free 2,6-dichloro-3- and -4-hydroxybenzonitrile, their glucuronide conjugates, ester glucuronides of the principal aromatic acids that are present in the hydrolysed urines, and two sulphur-containing metabolites analogous to mercapturic acids or premercapturic acids. 6. Prefix is thus extensively transformed into 2,6-dichlorobenzonitrile: R.CS.NH(2)-->R.CN+H(2)S, where R=C(6)H(3)Cl(2). However, the competitive reaction: R.CS.NH(2)+H(2)O-->R.CO.NH(2)+H(2)S takes place to a very limited extent.     

Isotope effects on binding of NAD+ to lactate dehydrogenase

The isotope effect on binding [4-2H]NAD+ and [4-3H]NAD+ to lactate dehydrogenase has been shown to be 1.10 +/- 0.03 by whole molecule isotope ratio mass spectrometry and 1.085 +/- 0.01 by 3H/14C scintillation counting. These values demonstrate that specific interactions of the nicotinamide ring with the enzyme make the C-H bond at C-4 less stiff in the binary complex.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

Comprehensive analysis of collagen metabolism in vitro using [4(3H)]/[14C]proline dual-labeling and polyacrylamide gel electrophoresis

A method to simultaneously quantify the production, secretion, and prolyl hydroxylation of individual types of collagen in cell culture samples has been developed. Collagens were biosynthetically labeled with a mixture of [14C]proline and [4-3H]proline. The labeled collagens were isolated and their component alpha-chains were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Migration of the collagen alpha-chains was determined by fluorography, and radioactivity in excised bands was quantified by scintillation counting. [14C]Proline labeling of collagen chains was used to determine the production and secretion of the different types of collagen. The ratios of the component alpha 1(I) and alpha 2(I) chains of type I collagen were also determined in this way. Prolyl hydroxylation of collagen alpha-chains was readily determined by measurement of their 3H:14C ratios. Following 4-hydroxylation, 3H was lost from the [4-3H]proline with alteration of this ratio. This dual-labeling method is suitable for the comprehensive analysis of collagen metabolism in multiple samples.     

Biosynthesis of trans,trans- and cis,trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.     

Metabolism of vitamin D. A new cholecalciferol metabolite, involving loss of hydrogen at C-1, in chick intestinal nuclei

1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4-(14)C]cholecalciferol and [1-(3)H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The (3)H/(14)C ratios in these four radioactive components were the same as that of the dosed material (4.7:1) with the exception of the most polar material. The (3)H/(14)C ratio was lower in the fourth, most polar, metabolite (0.4:1-1.8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0.5mug. of [4-(14)C,1-(3)H]cholecalciferol. This polar metabolite from the intestine also had the lowest (3)H/(14)C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of (14)C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered (3)H/(14)C ratio, in this case <0.2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered (3)H/(14)C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered (3)H/(14)C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of (3)H relative to (14)C are discussed in relation to possible chemical structures of this new polar metabolite.     

Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.     

Low background scintillation counting

Counting radioactive samples with Beckman Instrument's Ready Caps, using a restricted energy window, LL-UL = 400-1000, resulted in machine backgrounds of under 2 cpm and efficiencies of counting relative to liquid scintillation cocktails (LSC) of 51%, 65%, 57%, 62%, and 1% for 32P, 125I, 14C, 35S and 3H, respectively. Signal-to-noise ratios from a quantitative molecular hybridization technique were increased 8-10 fold. There may be a general application for this product in experiments yielding low amounts of radioactivity in liquid samples.     

Stereochemical determination of carbon partitioning between photosynthesis and photorespiration in C3 plants: use of (3R)-D-[3-3H1, 3-14C]glyceric acid

When (3R)-D-[3-3H1,3-14C]glyceric acid is supplied in tracer amounts to illuminated tobacco leaf discs, the acid penetrates to the chloroplasts without loss of 3H, and is phosphorylated there. Subsequent metabolism associated with the reductive photosynthetic cycle fully conserves 3H. Oxidation of ribulose bisphosphate (RuBP) by RuBP carboxylase-oxygenase (EC 4.1.1.39) results in the formation of (2R)-[2-3H1, 14C]glycolic acid which, on oxidation by glycolate oxidase (EC 1.1.3.1), releases 3H to water. Loss of 3H from the combined photosynthetic and photorespiratory systems is, therefore, associated with the oxidative photorespiratory loop. Assuming steady-state conditions and a basic metabolic model, the fraction of RuBP oxidized and the photorespiratory carbon flux relative to gross or net CO2 fixation can be calculated from the fraction of supplied 3H retained in the triose phosphates exported from the chloroplasts. This retention can be determined from the 3H:14C ratio for glucose obtained from isolated sucrose. The dependence of 3H retention upon O2 and CO2 concentrations can be deduced by assuming simple competitive kinetics for RuBP carboxylase-oxygenase. The experimental results confirmed the stereochemical assumptions made. Under conditions of negligible photorespiration 3H retention was essentially complete. The change in 3H retention with O2 and CO2 concentrations were investigated. For leaf discs (upper surface up) in normal air, it was estimated that 39% of the RuBP was oxidized, 32% of the fixed CO2 was photorespired, and the photorespiration rate was 46% of the net photosynthetic CO2 fixation rate. These are minimal estimates, as it is assumed that the only source of photorespired CO2 is glycine decarboxylation.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

Turnover and uptake of double-labelled high-density lipoprotein sphingomyelin in the adult rat

Rat HDL containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin was prepared by incubating labelled sphingomyelin liposomes with serum. HDL was then separated by ultracentrifugation and purified by gel-filtration chromatography. The maximum transfer was reached when 1.5 microliter sphingomyelin was incubated in the presence of 1 ml of serum at 37 degrees C for 1 h. When transfer was limited to a 5-7% increase in HDL mass, no significant change was observed in the HDL electrophoretic pattern, and rats could therefore be injected with this type of HDL under physiological conditions. Plasma radioactivity decay was followed for 24 h, and the recovery of both isotopes in 11 tissues was studied 24 h after the injection. The decay in plasma of both isotopes followed three exponential phases. During the first two phases, both isotopes disappeared with the same velocity (t1/2 = 12.8 and 98-105 min for the first and second phases, respectively). 10 h after injection, 3H had disappeared more slowly than 14C (t1/2 = 862 and 502 min for 3H and 14C, respectively) and 24 h after injection, only 1.5% of 14C and 2.5% of 3H remained in the plasma. This radioactivity was located mainly in HDL (80-85% for 3H and 14C), with a 3H/14C ratio close to that of injected sphingomyelin, and in VLDL, with the same isotopic ratio as that of liver lipids. Some 3H was associated with non-lipoprotein proteins. 17.5% of 3H and 23.4% of 14C were recovered in the liver, 1.6% of each isotope in erythrocytes, and 1.4% of 3H and 0.6% of 14C in kidney. Less than 1% of each isotope was recovered in each of the other tissues. Phosphatidylcholine was the lipid most labelled, and in several tissues sphingomyelin had a 3H/14C ratio close to that of injected sphingomyelin, showing an uptake without prior hydrolysis.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

A water-miscible, nonhazardous liquid scintillation cocktail

The optimal way to count aqueous samples by liquid scintillation counting is in a homogeneous solution. Technical limitations have previously made this difficult. Triton X-100 is a water-miscible liquid scintillant which counts ¹⁴C with 80% efficiency and ³H with 17% efficiency. It has a high flash point (over 300°F), is nonvolatile, and does not cause swelling or leaching when used in polyethylene vials. Liquid-scintillation counting cocktail using Triton X-100 as the sole scintillant (i.e., no toluene or xylene) does not have to be disposed of as a hazardous waste. The large aqueous sample capacity of a miscible cocktail, its safety, and ease of disposal make its use highly attractive for many applications.     

Comparison of 20-, 22-, and 24-carbon n-3 and n-6 polyunsaturated fatty acid utilization in differentiated rat brain astrocytes

Astrocytes convert n-6 fatty acids primarily to arachidonic acid (20:4n-6), whereas n-3 fatty acids are converted to docosapentaenoic (22:5n-3) and docosahexaenoic (22:6n-3) acids. The utilization of 20-, 22- and 24-carbon n-3 and n-6 fatty acids was compared in differentiated rat astrocytes to determine the metabolic basis for this difference. The astrocytes retained 81% of the arachidonic acid ([(3)H]20:4n-6) uptake and retroconverted 57% of the docosatetraenoic acid ([3-(14)C]22:4n-6) uptake to 20:4n-6. By contrast, 68% of the eicosapentaenoic acid ([(3)H]20:5n-3) uptake was elongated, and only 9% of the [3-(14)C]22:5n-3 uptake was retroconverted to 20:5n-3. Both tetracosapentaenoic acid ([3-(14)C]24:5n-3) and tetracosatetraenoic acid ([3-(14)C]24:4n-6) were converted to docosahexaenoic acid (22:6n-3) and 22:5n-6, respectively. Therefore, the difference in the n-3 and n-6 fatty acid products formed is due primarily to differences in the utilization of their 20- and 22-carbon intermediates. This metabolic difference probably contributes to the preferential accumulation of docosahexaenoic acid in the brain.