Whole body and tissue cholesterol turnover in the baboon

Cholesterol turnover was studied in four baboons by injecting [14C]cholesterol 186 days and [3H]cholesterol 4 days before necropsy, and fitting a two- or three-pool model to the resulting specific activity-time data. At necropsy, cholesterol mass and specific activity were determined for the total body (minus the central nervous system) and for many tissues. A pool model permits the estimation, from the plasma specific activity-time curve alone, of total body cholesterol within a limited range, depending upon the extent of side pool synthesis. The principal aim of this study was to estimate the extent of cholesterol synthesis in the side pools of the model, by computing the amount of side pool synthesis needed to equal the measured total body cholesterol. Central pool synthesis varied from 61 to 89% of the total cholesterol production rate. Thus, approximately 25% (11 to 39%) of the production rate arose from peripheral (pool 3 for the three-pool, and pool 2 for the two-pool model) cholesterol synthesis. Moreover, the finding that the measured total body cholesterol fell within the range obtained from the kinetic analysis by using reasonable assumptions (namely, that zero or that half the production rate occurred in the side pools), provides evidence for the physiological validity of the model. A second aim of this study was to explore cholesterol turnover in various tissues. A pool model predicts that rapidly turning over tissues will have higher specific activities at early times and lower specific activities at later times after injection of tracer relative to slowly turning over tissues, except where significant synthesis occurs. Tissues were ranked 1 to 17 for 3H and 17 to 1 for 14C cholesterol specific activity values. Except for the GI tract and testis, the tissues had similar ranks for both 3H and 14C, further validating model predictions. Results in all four baboons were similar. Turnover rates for the different tissues loosely fell into three groups which were turning over at fast, intermediate, and slow rates. Finally, the magnitude of variation of cholesterol specific activity was moderate for several distributed tissues (fat, muscle, arteries, and the alimentary tract), but was small for liver. Cholesterol turnover in serial biopsies of skin, muscle, and fat could, however, be fitted with a single pool to estimate tissue turnover rates.     

Human–Human Hybridomas from Lung Cancer Patients Secrete Anti-DNA Antibodies

The effects of modification of the arginine/lysine ratio of dietary protein on the cholesterol kinetics were studied in male rats. Single amino acids (lysine to soybean protein and arginine to casein) were added to approximate the arginine/lysine ratio in different proteins. After acclimation to these diets for 30 days, rats were administered intravenous [¹⁴C]cholesterol and oral [³H]cholesterol. Analysis of the die-away curve of [¹⁴C]cholesterol showed an apparent independence of cholesterol kinetics to the dietary manipulations, but there was a moderate reduction of the size of the slowly exchangeable pool and of the biliary concentration of cholesterol when lysine was added to soybean protein. Addition of amino acids neither influenced cholesterol absorption nor the fecal excretion of the radioactivities from labeled cholesterol. The results indicate that manipulating the arginine/lysine ratio of dietary protein by adding single amino acids is not necessarily effective in ameliorating cholesterol metabolism in rats, although the arginine addition caused a significant reduction of serum cholesterol and triglyceride.     

Cellular cholesterol efflux. Role of cell membrane kinetic pools and interaction with apolipoproteins AI, AII, and Cs.

Efflux of [14C]cholesterol from various cells was monitored in the presence of discoidal complexes of egg phosphatidylcholine and purified apolipoproteins, containing either apoAI, AII, or Cs. Particles containing apoAI were more efficient acceptors than those containing apoAII or Cs when the donor cells were J774 macrophages. No differences were observed when the same acceptor preparations were exposed to Fu5AH rat hepatoma or rabbit aortic smooth muscle cells. The differential efficiency of apolipoproteins in stimulating cholesterol removal from J774 cells was maintained in a plasma membrane-enriched fraction isolated from the same cells. Nonlinear regression analysis of kinetic data obtained from J774 cells exposed to apoAI complexes indicated that cholesterol efflux was best fitted to a curve describing the release from two kinetic compartments. Approximately 10% of cholesterol was transferred from a rapidly exchangeable pool with a t1/2 ranging between 1.5 and 3 h, and the remaining fraction was released from a slower pool with a t1/2 of about 20 h. Modulation of cholesterol efflux from J774 cells by either varying the concentration or the apolipoprotein composition of the acceptors influenced the size of the pools and the t1/2 of the slow pool. Kinetics of cholesterol efflux from membranes isolated from J774 cells also best fit a two-compartment model and modification of the apolipoprotein composition of the acceptor induced a pattern of changes in pool size and half-time similar to that described for whole cells. In the three cell lines studied, we consistently resolved a slow pool with a half-time ranging between 15 and 20 h. In smooth muscle cells only the slow pool was evident, whereas in Fu5AH a very large fast pool was also resolved. In contrast to J774 cells, apolipoprotein composition of the acceptor did not influence the pools in these two cell lines. These results led us to propose a new model regarding the influence of multiple kinetic pools of cholesterol on the regulation of cholesterol desorption from the cell membrane.     

Lysosomal membrane cholesterol dynamics

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.     

A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria

Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 +/- 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t(1/2) = 202 +/- 19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t(1/2) = 4.1 +/- 0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation.     

Turnover of carbon isotopes in tail hair and breath CO<Subscript>2</Subscript> of horses fed an isotopically varied diet

Temporal stable isotope records derived from animal tissues are increasingly studied to determine dietary and climatic histories. Despite this, the turnover times governing rates of isotope equilibration in specific tissues following a dietary isotope change are poorly known. The dietary isotope changes recorded in the hair and blood bicarbonate of two adult horses in this study are found to be successfully described by a model having three exponential isotope pools. For horse tail hair, the carbon isotope response observed following a dietary change from a C₃ to a C₄ grass was consistent with a pool having a very fast turnover rate (t _1/2~0.5 days) that made up ~41% of the isotope signal, a pool with an intermediate turnover rate (t _1/2 ~4 days) that comprised ~15% of the isotope signal, and a pool with very slow turnover rate (t _1/2 ~140 days) that made up ~44% of the total isotope signal. The carbon isotope signature of horse blood bicarbonate, in contrast, had a different isotopic composition, with ~67% of the isotope signal coming from a fast turnover pool (t _1/2 0.2 days), ~17% from a pool with an intermediate turnover rate (t _1/2 ~3 days) and ~16% from a pool with a slow turnover rate (t _1/2 ~50 days). The constituent isotope pools probably correspond to one exogenous and two endogenous sources. The exogenous source equates to our fast turnover pool, and the pools with intermediate and slow turnover rates are thought to derive from the turnover of metabolically active tissues and relatively inactive tissues within the body, respectively. It seems that a greater proportion of the amino acids available for hair synthesis come from endogenous sources compared to the compounds undergoing cellular catabolism in the body. Consequently, the isotope composition of blood bicarbonate appears to be much more responsive to dietary isotope changes, whereas the amino acids in the blood exhibit considerable isotopic inertia.     

Sterol carrier protein-2 alters high density lipoprotein-mediated cholesterol efflux

Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.     

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

Effect of infrequent feeding and increased physical activity on cholesterol kinetics in the rat.

The authors studied the effect of chronic physical exercise (running in a rotating drum at 850 m/hour, 5 times a week for 16 weeks) on the size of the cholesterol body pools and on cholesterol kinetics in adult male Wistar rats fed on a standard diet either ad libitum or 2 hours daily [33 weeks]. These data were obtained by mathematical analysis of the curve expressing the correlation of specific plasma cholesterol activity to time after a single dose of cholesterol-4-14C. Chronic physical stress and infrequent feeding, as separate experimental stimuli, both caused cholesterol to shift from the blood plasma at a higher rate and reduced the size of one or both cholesterol body pools (with quick or slow turnover, pools A and B). Physical exercise also reduced fractional cholesterol turnover in pool A. When the two stimuli were combined, i.e. in infrequently fed and chronically stressed rats, the rate of the cholesterol shift from the blood plasma slowed down, the total and irreversible shift of cholesterol from pool A diminished and the production rate in this pool also fell.     

Efflux of cholesterol from different cellular pools

Free cholesterol is very efficiently removed from cells by 2-hydroxypropyl-beta-cyclodextrins. The efflux of cholesterol occurs from two distinct kinetic pools: the half-times (t(1/2)) for the two pools in CHO-K1 cells are 15 +/- 5 s and 21 +/- 6 min and they represent 25% +/- 5% and 75% +/- 5% of the readily exchangeable cell cholesterol, respectively. In this study we have determined that the fast pool and the majority of the slow kinetic pool for cholesterol efflux are apparently present in the plasma membrane. Numerous agents that inhibit intracellular cholesterol trafficking are unable to affect either the size or the t(1/2) for efflux of either kinetic pool. In contrast, treatment of the cells with N-ethylmaleimide (NEM), exogenous lipases such as sphingomyelinase and phospholipase C, calcium ionophore A23187, or heat resulted in the dramatic increase in the size of the fast kinetic pool of cholesterol. These changes in the kinetics of cholesterol efflux are not specific to the nature of the extracellular acceptor indicating that they are a consequence of changes in the cell plasma membrane. The above treatments disrupt the normal organization of the lipids in the plasma membrane via either hydrolysis or randomization. The phosphatidylcholine and sphingomyelin present in the plasma membrane are critical for maintaining the two kinetic pools of cholesterol; any alteration in the amount or the location of these phospholipids results in an enhancement of efflux by redistributing cholesterol into the fast kinetic pool.     

Tissue distribution of phenoxybenzamine in the rat. Lack of adipose tissue storage

Rats were given the basic lipophilic drug, phenoxybenzamine, in single i.v. doses of 0.4 and 30 mg/kg. The drug was determined in plasma and 7 tissues by a new HPLC method. Adipose tissue reached peak levels after 30 minutes. At that time levels in heart, kidney, and brain were higher than in subcutaneous, epididymal, and mesenteric adipose tissues. The percentage metabolites in adipose tissue, kidney, and liver was 0, 88, and 96, respectively. Pretreatment with SKF 525-A inhibited metabolism and increased the tissue levels of unchanged drug, however, even under these conditions storage in adipose tissues did not occur, the adipose storage index being still less than unity. These results contradict findings of the 1950s which have been repeatedly reported in textbooks and reviews, but they confirm observations that basic drugs, even when highly lipophilic, are not being stored in adipose tissues.     

Interpretation of Tracer Data: Significance of the Number of Terms in Specific Activity Functions

It has already been shown that the number of pools in an open system in the steady state cannot be determined from the number of exponential terms in the specific activity function of a pool, even if the data were free from experimental error. However, some information is conveyed by the number of exponential terms. The information is different depending upon whether the data are obtained from the pool into which the tracer is introduced or from another pool. In the latter case, the number of exponential terms is shown to indicate the maximum number of intermediate pools involved in the shortest path of transfer of material from the injected pool to the pool in question. With regard to the former case, this paper is restricted to functions with two exponential terms and shows which systems of n pools (n >/= 2) are consistent with such data. Consequently, biexponential experimental curves can be interpreted in terms of models consisting of an unrestricted number of pools in which each pool is defined in terms of fast mixing. The generalization to cases of functions with more than two exponential terms can be carried out in a similar manner.     

Mixing rate of phosphate between plasma and interstitial body fluid of cows

Radiophosphate was injected into the left jugular vein of dairy cows. Blood samples were taken frequently from the right jugular vein during the first hour after injection. Between 20 minutes and 1 hour after injection, the decrease in plasma radioactivity could be formulated as a first order process, designated as "process 3," with a turnover time of 50 minutes. From 5 to 20 minutes after injection the decrease in plasma activity could be interpreted as the result of mixing plasma phosphate with another phosphate pool, designated as the second pool. The capacity of this second pool was derived as a constant in a kinetic equation, so chosen that the resulting mixing rates were independent of time. For two cows the capacity of the second pool was 5 and 8 times, respectively. the phosphate content of the plasma. This result led to the working hypothesis that the major part of the second pool was the phosphate in the interstitial tissue fluid. The turnover time of the plasma phosphate in the mixing process with the second pool amounted to an average of 14 minutes for 5 lactating cows, and an average of 21 minutes for 2 dry cows. This result was obtained under the assumption that the slow first order process 3 is parallel to the mixing process. The assumption that the slower first order process is in series with the mixing process reduces the resulting mixing time to about four-fifths of that reported above. The calculation of process 2 which deviates from first order may be applicable to numerous turnover processes in which both exchange pools have a limited capacity.     

Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor signaling

T lymphocytes contain two kinetic pools of cholesterol extractable with methyl-beta-cyclodextrin (m-beta-CD): a fast pool (31.5%, t1/2=17 s) and a slow pool (68.5%, t1/2=15 min). Purification of detergent-resistant membranes (DRMs) shows that the fast pool corresponds to buoyant cholesterol. Cholesterol extraction of the fast pool (i.e. cholesterol from rafts) still allows the buoyancy of signaling proteins and their phosphorylation under CD3 stimulation. Cholesterol depletion of the slow pool (i.e. cholesterol from membranes other than rafts) is accompanied by the extraction of the whole raft followed by the inhibition of CD3-induced tyrosine-phosphorylations. Cholesterol oxidase (COase) allows a specific oxidation of raft cholesterol into cholestenone. Cholestenone leaves the DRMs and accumulates as Triton X-100-soluble material. Specific cholesterol-rich raft disruption by COase does not inhibit the activation of either Jurkat cells or T CD4+ lymphocytes. Our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that a cholesterol-poor subtype of rafts is involved in signal transmission via the TCR.     

Fate of exogenous and newly synthesized cholesterol in intestinal cell lines.

1. The current study was undertaken to test the existence of functionally distinct intracellular pools of cholesterol depending on the origin: neosynthesis or exogenous. 2. This was performed on two subpopulations, either differentiated or undifferentiated, of the HT29 cell line. 3. A parallel study was also carried out on Caco-2 cells. 4. First we checked the ability of differentiated HT29 cells to secrete lipids into the medium and found that lipid production was efficient but less so than in Caco-2 cells. 5. In contrast, undifferentiated HT29 cells were unable to secrete lipids into the medium. 6. Then we studied the fate of [14C]cholesterol incorporated into micellar preparations and of [14C]mevalonate in the different models. 7. The data obtained with labelled exogenous cholesterol show that it enters the membrane cholesterol pool as well as, for the differentiated models, the cholesteryl ester pool. 8. Similarly, labelled newly synthesized cholesterol could be used for membrane formation as well as for incorporation into cholesteryl esters. 9. Thus, in HT29 subpopulations as well as in Caco-2 cells, the results suggest the existence of a common pool of cholesterol whatever its origin.     

GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria

Pregnenolone synthesis from cholesterol by adrenal mitochondria isolated from ether-stressed rats exhibits a biphasic time course: upon the addition of a reducing substrate (e.g. malate), a rapid phase of pregnenolone formation occurs during the first 5 min, which has been interpreted as the metabolism of a steroidogenic pool of cholesterol, probably in the inner membrane. A slower rate follows, which is interpreted as translocation of cholesterol into the steroidogenic pool. While a 30-min preincubation of mitochondria with cholesterol alone did not affect the extent of the rapid phase, preincubation with GTP plus cholesterol extended the first phase, resulting in an up to 2-fold increase in pregnenolone synthesis by 20-30 min. The apparent Km for GTP was 0.1-0.4 mM, and stimulation was maximal with preincubation times of 10-30 min, depending upon incubation conditions. Exogenous cholesterol was not required to observe a stimulatory effect, indicating that GTP reorganizes the endogenous mitochondrial cholesterol pools. Nevertheless, stimulation was greater when exogenous cholesterol was provided, consistent with enhanced utilization of both endogenous and exogenous cholesterol. Stimulation by GTP was also seen in mitochondria isolated from cycloheximide-injected/ether-stressed rats, although the activity in these preparations was always lower than that in mitochondria from ether-stressed rats. The stimulation was specific for GTP, since many other nucleotides (e.g. ATP, GDP, and ITP) and GTP analogues (guanosine 5'-O-(3-thiotriphosphate and guanosine 5'-(beta,gamma-imino)triphosphate) had no effect. The GTP-activated state was reversible: after GTP hydrolysis by a mitochondrial GTPase, pregnenolone synthesis returned to the basal level. Sonic disruption of mitochondria abolished the stimulatory effect of GTP. These results suggest that GTP enhances pregnenolone synthesis by promoting the movement of cholesterol to the steroidogenic pool, consistent with a recently proposed general role for GTP in some vectorial transport processes (Bourne, H. R. (1988) Cell 53, 669-671).     

Placental transfer of cholesterol-4-14C into rabbit and guinea pig fetus

A tracer dose of cholesterol-4-(14)C was given daily in the diet of six pregnant guinea pigs to establish an isotopic steady state. At the time of parturition, maternal and fetal blood and fetal tissues were collected and analyzed for cholesterol content and cholesterol specific activity. A comparison of these specific activities in neonatal and maternal serum indicated that about 22% of the fetal serum cholesterol was transferred from maternal blood. In the newborn, tissues generally had the same cholesterol specific activity as serum. Brain tissue was an exception in having a specific activity only 8.4% of that of serum. Dietary cholesterol did not increase serum cholesterol levels in the newborn but did increase the percentage of fetal cholesterol derived from the maternal circulation. The rapid transfer of cholesterol-4-(14)C across the placenta was indicated by the appearance of this isotope in the newborn 2 days after its administration to pregnant rabbits. A considerable amount of the cholesterol content of newborn guinea pigs and rabbits originated from the maternal blood.     

Monoclonal Antibody-Mediated Cytotoxicity of Adipocytes

Monoclonal antibodies (MAbs) raised against porcine adipocyte plasma membranes were used to demonstrate complement-mediated cytotoxicity of adipocytes and preadipocytes in primary stromal-vascular (SV) cultures. Five of the six MAbs tested significantly reduced the number of fat cell clusters in cultures maintained in medium supplemented with pig serum and dexamethasone (PS/DEX) but not in cultures supplemented with insulin, transferrin, and selenium (ITS). Neither MAb nor complement alone affected fat cell cluster number. Treatment of both ITS and PS/DEX cultures with pools of 2 or more MAbs, in combination with complement, eliminated fat cell clusters in all instances. Treatment of cultures prior to appearance of cells containing lipid demonstrated that preadipocytes, or adipose lineage cells, could also be eliminated by MAb/complement treatment. Finally, injection of young rats with a pool of three of the MAbs produced a 30% reduction in inguinal fat pad weight without affecting other tissues. Adipocyte/ preadipocyte-depleted cultures can now be used as a model system to examine progression of cells through the adipose cell lineage at a time not previously possible with primary cells.     

Some remarks on the presence of organic phosphates in sediments

This article describes a new method developed to assess the size and nature of the organic phosphate pool. Using sediment suspensions from the Rhone, Garonne and Po rivers, inorganic P compounds, Fe(OOH) and CaCO₃ were removed using mild extractants at sediment pH. The residual phosphate was then fractionated into an acid soluble organic phosphate pool and a residual organic phosphate pool by acid hydrolysis (0.5 M H⁺). Both pools were quantitatively important, accounting for between 16 and 54% and 16 and 51% of total phosphate respectively. Acid hydrolysis was chosen since it yielded a distinct plateau, with high reproducibility, within 30 minutes.This fractionation permits a further study of dynamics and bioavailability of sediment org-P, without interference of Fe(OOH) and CaCO₃.In many studies in which changes in the organic pool were examined after extraction of inorganic phosphate, 0.5 M HCl was used to extract apatite bound phosphate. The results presented here show that this is likely to result in a considerable underestimation of the organic phosphate pool.