Impact of endothelial lipase on cellular lipid composition

Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [¹⁴C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [¹⁴C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [¹⁴C]-LPC accumulated in HAEC as well. Its conversion to [¹⁴C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [³H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.     

The biosynthesis of lubimin from [1-14C]isopentenyl pyrophosphate by cell-free extracts of potato tuber tissue inoculated with an elicitor preparation from Phytophthora infestans

A cell-free enzyme system, which catalyses the incorporation of radiolabel from [$\text{1}\text{2}$-¹⁴C]isopentenyl pyrophosphate into the sesquiterpenoid phytoalexin lubimin, has been prepared from tuber tissue of Solanum tuberosum inoculated with an elicitor preparation from Phytophthora infestans. Biosynthesis of lubimin is optimum at pH 7.$\text{3}\text{2}$-7.5 and is dependent upon Mg²⁺ and NADPH. Lubimin labelling by cell-free enzyme system prepared from tissue 48 hr after treatment with elicitor rises rapidly to a maximum over the first 30 min of incubation and does not decline for a further 150 min. The biosynthetic capacity for lubimin in cell free extracts can be observed as early as 3 hr after inoculation of tuber tissue, and rises to a maximum at about 48 hr after treatment, declining thereafter. Lubimin labelling is inhibited by iodoacetamide, the effect of which is reversed by 3,3-dimethylallylpyrophosphate. Preliminary observations on the cell-free system show that it will also catalyse the incorporation of [2-¹⁴C]mevalonic acid into lubimin in the presence ofan ATP-generating system.     

Gentamicin-induced alterations in phospholipid metabolism in cultured human proximal tubular cells

The effects of gentamicin on phospholipid levels and metabolism and the uptake of phosphatidylcholine (PC) adsorbed to low-density lipoprotein (LDL) were investigated in cultured human proximal tubular (PT) cells. Cells incubated with gentamicin (0.3 mM) for one to 21 days had a similar increase in the cell number and protein as compared to control cells. However, the cellular levels of phosphatidylcholine (PC) and sphingomyelin (SM), but not other phospholipids, increased in a time-dependent manner. Incubation of gentamicin (0.3 to 3.0 mM) resulted in a concentration-dependent increase in the cellular levels of PC (50% to 320%) and SM (20% to 40%). Gentamicin stimulated the incorporation of [¹⁴C]-acetate into diacylglycerol, PC, and SM in the order of 300%, 66%, and 20%, respectively, but not into lysophosphatidylcholine (LPC). Similarly, gentamicin stimulated the incorporation of [¹⁴C]-choline into PC and SM in the order of 300% and 172%, respectively, but not into LPC as compared to control cells. In addition, gentamicin also stimulated the incorporation of [¹⁴C]-choline into cytidine diphosphocholine (CDP-choline). However, the endocytosis of [¹⁴C]-PC-LDL was lower in cells incubated with gentamicin than in control cells. Thus, exogenously derived PC on LDL does not contribute to the increased cellular levels of PC in PT cells incubated with gentamicin. The activity of cytidine triphosphate (CTP):phosphocholine cytidyltransferase was moderately lower in cells incubated with gentamicin as compared to control. By contrast, the activity of phospholipase A₁ and phospholipase C was twofold lower in cells incubated with gentamicin for 21 days as compared to control. Thus, increased incorporation of [¹⁴C]-acetate and [¹⁴C]-choline into PC in cells incubated with gentamicin may not only be due to increased endogenous synthesis but to decreased catabolism of newly synthesized PC. We conclude that gentamicin impairs the lysosomal catabolism of PC, leading to its accumulation in PT cells. This phenomenon may be an indication of gentamicin-induced nephrotoxicity in man.     

Desaturation of oleate-[14C] in leaves of barley

Etiolated barley leaves when exposed to light desaturate oleate-[¹⁴C] to linoleate. The production of substantial amounts of radioactive linolenate was found only in very young, tightly rolled leaves. In oleate-[¹⁴C] pulse experiments, radioactive linolenate first appeared in phosphatidylcholine (PC) and only after a lag period did it begin to accumulate in monogalactosyldiacylglycerol (MGDG). The results indicate that in young, immature barley leaves linolenate is synthesized from oleate on the parent lipid, PC, and is then transferred to MGDG.     

Synthesis of molecular species of phosphatidylcholine and phosphatidylethanolamine by germinating soya bean

The composition and metabolism of phosphatidylcholines and phosphatidylethanomines of germinating soya bean Glycine max have been examined. Both phospholipids have a very similar fatty acid composition and distribution, with saturated acids located at the 1- position. The fatty acid composition and relative amounts of individual molecular species of the two phospholipids were also very similar. The relative amounts of the species were in the order tetraenoic pentaenoic trienoic = dienoic = monoenoic. In contrast, the labelling of the molecular species from choline Me[¹⁴C] or ethanolamine [2-¹⁴C] showed considerable differences. Phosphatidylethanolamine-[¹⁴C] showed 58% label in trienoic, 17% in tetraenoic, 18% in pentaneoic and 5% in dienoic species 48 hr after germination. The equivalent figures for phosphatidylcholine-[¹⁴C] were 37, 34, 13 and 15% respectively. An increase in labelling of the more unsaturated species was seen with time.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

The fate of 2,4,6-tri-(3′,5′-di-tert.-butyl-4′- hydroxybenzyl)mesitylene (Ionox 330) in the dog and rat

1. Unchanged Ionox 330 is quantitatively eliminated in the faeces of dogs, rats and man after oral administration, and ¹⁴C is absent from the urine and expired gases of rats intubated with [¹⁴C]Ionox 330. Dogs and rats do not show a sex difference in this pattern of elimination. 2. Quantitative elimination of [¹⁴C]Ionox 330 and the absence of ¹⁴C in the carcass and viscera of rats 72hr. after dosage show that this substance does not accumulate in the body. 3. No metabolites are formed in consequence of the ingestion of Ionox 330. 4. Rats eliminate three-quarters or more of a dose (285·7mg./kg. body wt.) of Ionox 330 in 24hr. and the remainder during 24–48hr., and dogs eliminate the whole dose (90mg./kg. body wt.) within 48hr. and a variable proportion within 24hr. These rates of elimination are consistent with the passage of unabsorbed material through the alimentary canal. 5. After removal of the alimentary canal, radioactivity is absent from the carcass and remaining viscera of rats 8, 16 and 24hr. after ingestion of [¹⁴C]Ionox 330, and this strongly suggests the absence of alimentary absorption. 6. The absence of ¹⁴C in the 24hr. bile of animals with biliary fistulae establishes that [¹⁴C]Ionox 330 is not absorbed from the gastro-intestinal tract.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

An improved toluene-triton-based liquid scintillation system for counting 14C-labeled compounds at ambient temperature

With respect to counting rate and stability, the standard toluene/Triton X-100 (2:1, v/v) scintillation system was neither adequate for assaying trichloro[¹⁴C]acetic acid in ethanol solution or in ethanol extracts from shoots and roots of wheat seedlings, nor appropriate for counting [¹⁴C]dicamba in ethanol extracts from roots of barley and oats seedlings. The counting rates decreased rapidly during the first 10 hr, followed by a further decline at slower rates. The addition of NCS (3.3%, v/v) made the system suitable for measuring a number of ¹⁴C-labeled compounds (3-amino-s-triazole, 2,4-dichlorophenoxyacetic acid, 3,6-dichloro-o-anisic acid, [(4-chloro-o-tolyl)oxy]acetic acid, and trichloroacetic acid) either dissolved in ethanol or extracted from seedlings of cereal crops.     

Soluble and particulate lysophospholipase in the aleurone and endosperm of germinating barley

Lysophospholipase was measured in extracts of germinating barley by determining the amount of free [¹⁴C]palmitate released from [1-¹⁴C] 1-palmitoyl-lysophosphatidylcholine (LPC). Soluble and particulate lysophospholipase activity was measured at 1-day intervals in extracts from the aleurone and endosperm of barley seeds germinated for 8 days. The soluble and particulate activities of the aleurone increase approximately in parallel with one another and after 8 days of germination have 20–30 times more activity than at day 1. The activity profiles and the distribution of the activity between the soluble and particulate forms of lysophospholipase in the endosperm are markedly different. With the exception of the first 2 days when the aleurone activity is low, the endosperm activity is less than that associated with the aleurone. The soluble activity increases during the first 3 days and is more active than that of the aleurone. Thereafter it diminishes and remains low. The particulate enzyme, however, increases dramatically between days 4 and 5 and remains moderately high. The fourth and fifth day represent that stage of germination when starch-bound LPC is released in concert with the increase in amylase activity. It is proposed that it is this particulate form of the endosperm activity which may be responsible for maintaining the level of free LPC low in the endosperm of the germinating seed.     

The effect of hypolipidemic drugs WY14643 and DH990, and lysophospholipids on the metabolism of oleate in plants

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [¹⁴C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [¹⁴C]oleate or [¹⁴C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [¹⁴C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.     

Differential responses of a range of photosynthetic tissues to a substituted pyridazinone,sandoz 9785. Specific effects on fatty acid desaturation

The effect of a substituted pyridazinone (4-chloro-5(dimethylamino)-2-phenyl-3(2H)pyridazinone; Sandoz 9785; BASF 13-338) on the formation of fatty acids from radiolabelled precursors has been studied in a number of angiosperms, bryophytes and algae. The labelling of [¹⁴C]linolenic acid was decreased by the herbicide in leaves of barley and rye grass and in cucumber cotyledons regardless of whether [¹⁴C]acetate,[¹⁴C]oleate or [¹⁴C]linoleate was used as precursor. A commensurate increase in the labelling of [¹⁴C]linoleic acid was also observed in these species. In contrast, the pattern of fatty acid labelling in maize, pea and spinach leaves was unaffected by 0.1 mM Sandoz 9785. More generalized inhibition of the incorporation of radioactivity from [¹⁴C]acetate into the fatty acids of bryophytes and algae was seen. Sandoz 9785 did not alter the distribution of radioactivity in different lipid classes of higher plant leaves, nor did it change the proportions of radioactive fatty ac ids in phosphatidylcholine. In contrast to phosphatidylcholine, which never contained more than trace amounts of [¹⁴C]linolenate, diacylgalactosylglycerol contained high levels of the radioactive acid. The relative labelling of linolenate was severely reduced in diacylgalactosylglycerol by Sandoz 9785 in sensitive angiosperms. Uptake studies, in which [³H]Sandoz 9785 was employed demonstrated that the uptake of Sandoz 9785 was reflection of water uptake. Following its uptake, Sandoz 9785 was rapidly converted into other compounds in pea but only gradually metabolized in cucumber and ryegrass. The results are interpreted as showing, firstly, that the different sensitivity of higher plants to Sandoz 9785 is due to variations both in uptake and in metabolism. Secondly, Sandoz 9785 specifically inhibits the desaturation of linoleate to linolenate and, thirdly, diacylgalactosylglycerol plays a role in this conversion.     

The nature of formate metabolism in greening barley leaves

The metabolism of [³H]formate has been examined in etiolated and greening leaves of barley (Hordeum vulgare), dwarf bean (Phaseolus vulgarls), broad bean (Vicia faba) and corn (Zea mays). Tritium was extensively incorporated by primary leaves incubated for 20-min periods in light or dark. The organic acids and free amino acids were the principal products of formate metabolism but these and other products were more heavily labelled in green tissues. Time course experiments with barley leaves revealed a rapid labelling of serine, accompanied by increasing amounts of ³H in glycine and aspartate as the feeding period was extended. These amino acid products were formed throughout a 4-day greening period with an approximate doubling in total incorporation being due to large accumulations of tritiated glycine and aspartate. The involvement of tetrahydrofolate-dependent reactions in formate metabolism was indicated by inhibition of [¹⁴C] and [³H]formate incorporation by the folate antagonist, aminopterin. Labelling of glycine and serine was also strongly inhibited (up to 90%) when the leaves were incubated with increasing concentrations of isonicotinylhydrazide.     

Polyprenyl quinones and α-tocopherol in Calendula officinalis

In various cellular subfractions of Calendula officinalis leaves a study was made of the distribution of polyprenyl quinones and α-tocopherol and the dynamics of their labelling with ¹⁴CO₂ and acetate-[1-¹⁴C] and incorporation of mevalonate-[2-¹⁴C] after 3 hr. It was confirmed that plastoquinone occurs only in the chloroplasts, ubiquinone only in the mitochondria and α-tocopherol in both these subfractions. Phylloquinone was found in the chloroplast and mitochondrial fractions as well as in the post-mitochondrial supernatant. Studies of the dynamics of radioactive precursor incorporation indicated that α-tocopherol is metabolized more rapidly than the polyprenyl quinones studied; the incorporation of mevalonate-[2-¹⁴C] suggests that the side chain of plastoquinone can be synthesized in the cytoplasm and transported to the chloroplasts.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Sitosterol biosynthesis in Hordeum vulgare

Excised barley embryos cultured on a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly biosynthesized sterols. Two deuterium atoms were present in 24-methylenecycloartanol, 24-methylenelophenol and campesterol and a maximum of four deuterium atoms were incorporated into 24-ethylidenelophenol, stigmasterol and sitosterol. Mevalonic acid-[2-¹⁴C(4R)4-³H₁] was utilized by the barley embryos to give 28-isofucosterol with a ³H-¹⁴C atomic ratio of 3:5 and stigmasterol and sitosterol with a ³H-¹⁴C atomic ratio of 2:5. 24-Methylenelophenol and 24-ethylidenelophenol were isolated from barley seed and 24-ethylidenelophenol-[2,4-³H₃] was incorporated into sitosterol by barley seedlings. These results show that in the production of sitosterol a 24-ethylidenesterol intermediate is produced and it is suggested that this is isomerized to give a Δ^24,(25) sterol prior to reduction to the saturated C₂₉ sterol side chain.     

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1-14C]oleate to individual phospholipids of platelets

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-¹⁴C]oleate resulting in esterification of [1-¹⁴C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-¹⁴C]oleate and esterification of [1-¹⁴C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 μM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 μM 2′,5′ dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM chloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.     

Metabolic studies of rat liver plasma membranes using d-[l-C]glucosamine

The metabolism of plama membranes of rat liver cells was studied using d-[l-¹⁴C]glucosamine. The labelling of plasma membranes occurred more slowly than that of microsomes, reaching a maximum at about 3 h after injection compared to 1.5 h for microsomes, and the radioactivity decayed with a half-life of 37 h which is close to the value obtained using [guanidino-¹⁴C]arginine to label proteins. Hexosamine and sialic acid of plasma membranes were found to metabolize at practically equal rates.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

STUDIES WITH MURINE LPC-1 PLASMACYTOMA USING [6-14C]ARGININE

[6-¹⁴C]Arginine ([6-¹⁴C]Arg) was used as an in vivo pulse label to study BALB/c murine LPC-1 plasmacytoma synthesis and secretion of its tumour-associated M component (IgG2a, k). With this isotope, an eight- to ten-fold enhancement in the labelling of the γ globulin region and ten-fold reduction in the albumin labelling were observed. Production and secretion of the M component was detected (within 30 min) after cell transfer. Only mice which received tumour cells showed significant labelling in the γ globulin region 24 hr after isotope injection. The labelling behaviour of the tumour M component correlated with the administered cell dose. The peak heights of radioactivity in the γ region increased with increments in cell number. When the percentage radioactivity diverted into M component was plotted as a function of cell dose, a linear relationship was noted. This study demonstrates the feasibility of using [6-¹⁴C]Arg as a tool to follow the newly synthesized tumour-associated protein, and provides a means of estimating tumour cell number.