In Vivo Effectiveness of Orlistat in the Suppression of Human Colorectal Cancer Cell Proliferation.

BACKGROUND/AIM: Fatty acid synthase (FASN) provides palmitate for cell membrane formation in colorectal cancer (CRC) cells, however, palmitate is also available in the blood of CRC patients. The aim of this study was to examine whether orlistat, a FASN inhibitor, is able to attenuate CRC cell growth despite the availability of extracellular palmitate. MATERIALS AND METHODS: Palmitate concentrations were measured in serum from CRC patients and healthy controls. HT-29 CRC cells were treated with orlistat and palmitate. RESULTS: Treatment of CRC cells with orlistat caused a dose-dependent inhibition of cell proliferation. In turn, delivery of extracellular palmitate at doses lower than those found in the serum of CRC patients reversed inhibition by orlistat concentrations of up to 10 µM. CONCLUSION: Inhibition of CRC cell proliferation by orlistat is reversed by palmitate which is present at high levels in the serum. Therefore, orlistat may be effective in vivo only at high concentrations.

Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge.

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Intramyocellular Ceramides: Subcellular Concentrations and Fractional De Novo Synthesis in Postabsorptive Humans.

We investigated the relationship between insulin resistance markers and subsarcolemmal (SS) and intramyofibrillar (IMF) ceramide concentrations, as well as the contribution of plasma palmitate (6.5-h infusion of [U-13C]palmitate) to intramyocellular ceramides. Seventy-six postabsorptive men and women had muscle biopsies 1.5, 6.5, and 24 h after starting the tracer infusion. Concentrations and enrichment of muscle ceramides were measured by liquid chromatography-tandem mass spectrometry. We found that HOMA of insulin resistance, plasma insulin, and triglyceride concentrations were positively correlated with SS C16:0 and C18:1 ceramide, but not SS C14:0-Cer, C20:0-Cer, C24:0-Cer, and C24:1-Cer concentrations; IMF ceramide concentrations were not correlated with any metabolic parameters. The fractional contribution of plasma palmitate to 16:0 ceramide was greater in SS than IMF (SS, 18.2% vs. IMF, 8.7%; P = 0.0006). Plasma insulin concentrations correlated positively with the fractional contribution of plasma palmitate to SS 16:0 ceramide. The fractional contribution of plasma palmitate to intramyocellular SS 16:0 ceramide was positively correlated with SS C16:0 ceramide concentrations (γ = 0.435; P = 0.002). We conclude that skeletal muscle SS ceramides, especially C16 to C18 chain lengths and the de novo synthesis of intramyocellular ceramide from plasma palmitate are associated with markers of insulin resistance.

Restoring redox balance enhances contractility in heart trabeculae from type 2 diabetic rats exposed to high glucose.

Hearts from type 2 diabetic (T2DM) subjects are chronically subjected to hyperglycemia and hyperlipidemia, both thought to contribute to oxidizing conditions and contractile dysfunction. How redox alterations and contractility interrelate, ultimately diminishing T2DM heart function, remains poorly understood. Herein we tested whether the fatty acid palmitate (Palm), in addition to its energetic contribution, rescues function by improving redox [glutathione (GSH), NAD(P)H, less oxidative stress] in T2DM rat heart trabeculae subjected to high glucose. Using cardiac trabeculae from Zucker Diabetic Fatty (ZDF) rats, we assessed the impact of low glucose (EG) and high glucose (HG), in absence or presence of Palm or insulin, on force development, energetics, and redox responses. We found that in EG ZDF and lean trabeculae displayed similar contractile work, yield of contractile work (Ycw), representing the ratio of force time integral over rate of O2 consumption. Conversely, HG had a negative impact on Ycw, whereas Palm, but not insulin, completely prevented contractile loss. This effect was associated with higher GSH, less oxidative stress, and augmented matrix GSH/thioredoxin (Trx) in ZDF mitochondria. Restoration of myocardial redox with GSH ethyl ester also rescued ZDF contractile function in HG, independently from Palm. These results support the idea that maintained redox balance, via increased GSH and Trx antioxidant activities to resist oxidative stress, is an essential protective response of the diabetic heart to keep contractile function.

Relative oral bioavailability of glycidol from glycidyl fatty acid esters in rats.

In order to quantify the relative bioavailability of glycidol from glycidyl fatty acid esters in vivo, glycidyl palmitoyl ester and glycidol were orally applied to rats in equimolar doses. The time courses of the amounts of glycidol binding to hemoglobin as well as the excretion of 2,3-dihydroxypropyl mercapturic acids were determined. The results indicate that glycidol is released from the glycidyl ester by hydrolysis and rapidly distributed in the organism. In relation to glycidol, there was only a small timely delay in the binding to hemoglobin for the glycidol moiety released from the ester which may be certainly attributed to enzymatic hydrolysis. In both cases, however, an analogous plateau was observed representing similar amounts of hemoglobin binding. With regard to the urinary excretion of mercapturic acids, also similar amounts of dihydroxypropyl mercapturic acids could be detected. In an ADME test using a virtual double tag (³H, ¹4C) of glycidyl palmitoyl ester, a diverging isotope distribution was detected. The kinetics of the ¹4C-activity reflected the kinetics of free glycidol released after hydrolysis of the palmitoyl ester. In view of this experimental data obtained in rats, it is at present justified for the purpose of risk assessment to assume complete hydrolysis of the glycidyl ester in the gastrointestinal tract. Therefore, assessment of human exposure to glycidyl fatty acid ester should be regarded as an exposure to the same molar quantity of glycidol.

Lipidization of human interferon-alpha: a new approach toward improving the delivery of protein drugs.

Human interferon-alpha (IFN-alpha), a 19.2 KD protein containing two disulfide bonds (cys1-cys98; cys29-cys138), was reduced and modified with a reversible lipidization agent. The product of the lipidization, PAL-IFN, was homogenous, with four palmitoyl moieties linked to the four Cys residues in the protein molecule via reversible disulfide linkages. The far-UV circular dichroism (CD) spectrum of PAL-IFN was virtually overlapped with that of IFN, indicating that the IFN structure was not altered by the modification. After iv injection in mice of 0.1 mg/kg of PAL-IFN, a low level of serum IFN activity was sustained for more than 8 h, while serum IFN activity was rapidly diminished to an undetectable level at 2 h post IFN injection at the same dose. Evidence suggested that IFN was slowly released from PAL-IFN into blood circulation upon reduction of the disulfide bonds in vivo. Furthermore, the liver-targeting effect of PAL-IFN was demonstrated by the observation that the level of 2'-5' oligoadenylate synthetase (OAS) expressed in the liver of mice treated with PAL-IFN was significantly higher than that with IFN. In conclusion, reversible lipidization can potentially achieve both a prolonged plasma half-life and an enhanced liver-targeting effect of IFN in antiviral therapy.

Tumor necrosis factor-alpha modulates human in vivo lipolysis.

CONTEXT: Low-grade systemic inflammation is a feature of most lifestyle-related chronic diseases. Enhanced TNF-alpha concentrations have been implicated in the development of hyperlipidemia. OBJECTIVE: We hypothesized that an acute elevation of TNF-alpha in plasma would cause an increase in lipolysis, increasing circulatory free fatty acid (FFA) levels. SUBJECTS AND METHODS: Using a randomized controlled, crossover design, healthy young male individuals (n = 10) received recombinant human (rh) TNF-alpha (700 ng/m(-2).h(-1)) for 4 h, and energy metabolism was evaluated using a combination of tracer dilution methodology and arterial-venous differences over the leg. RESULTS: Plasma TNF-alpha levels increased from 0.7 +/- 0.04 to 16.7 +/- 1.8 pg/ml, and plasma IL-6 increased from 1.0 +/- 0.2 to 9.2 +/- 1.0 pg/ml (P < 0.05) after 4-h rhTNF-alpha infusion. Here, we demonstrate that 4-h rhTNF-alpha infusion increases whole body lipolysis by 40% (P < 0.05) with a concomitant increase in FFA clearance, with no changes in skeletal muscle FFA uptake, release, or oxidation. Of note, systemic glucose turnover and lactate and catecholamine levels were unaffected by rhTNF-alpha infusion. CONCLUSION: This study demonstrates that a relatively low dose of rhTNF-alpha induces systemic lipolysis and that the skeletal muscle fat metabolism is unaffected.

Inhibition of lipolysis stimulates peripheral glucose uptake but has no effect on endogenous glucose production in HIV lipodystrophy.

HIV-infected patients with lipodystrophy (HIV lipodystrophy) are insulin resistant and have elevated plasma free fatty acid (FFA) concentrations. We aimed to explore the mechanisms underlying FFA-induced insulin resistance in patients with HIV lipodystrophy. Using a randomized, placebo-controlled, cross-over design, we studied the effects of an overnight acipimox-induced suppression of FFAs on glucose and FFA metabolism by using stable isotope-labeled tracer techniques during basal conditions and a two-stage euglycemic-hyperinsulinemic clamp (20 and 50 mU insulin/m(2) per min, respectively) in nine patients with nondiabetic HIV lipodystrophy. All patients received antiretroviral therapy. Biopsies from the vastus lateralis muscle were obtained during each stage of the clamp. Acipimox treatment reduced basal FFA rate of appearance by 68.9% (95% CI 52.6-79.5) and decreased plasma FFA concentration by 51.6% (42.0-58.9) (both, P < 0.0001). Endogenous glucose production was not influenced by acipimox. During the clamp, the increase in glucose uptake was significantly greater after acipimox treatment compared with placebo (acipimox: 26.85 micromol x kg(-1) x min(-1) [18.09-39.86] vs. placebo: 20.30 micromol x kg(-1) x min(-1) [13.67-30.13]; P < 0.01). Insulin increased phosphorylation of Akt Thr(308) and glycogen synthase kinase-3beta Ser(9), decreased phosphorylation of glycogen synthase (GS) site 3a + b, and increased GS activity (percent I-form) in skeletal muscle (P < 0.01). Acipimox decreased phosphorylation of GS (site 3a + b) (P < 0.02) and increased GS activity (P < 0.01) in muscle. The present study provides direct evidence that suppression of lipolysis in patients with HIV lipodystrophy improves insulin-stimulated peripheral glucose uptake. The increased glucose uptake may in part be explained by increased dephosphorylation of GS (site 3a + b), resulting in increased GS activity.

No effect of menstrual cycle phase on basal very-low-density lipoprotein triglyceride and apolipoprotein B-100 kinetics.

Dyslipidemia, manifested by increased plasma triglyceride (TG), increased total and LDL-cholesterol concentrations and decreased HDL-cholesterol concentration, is an important risk factor for cardiovascular disease. Premenopausal women have a less atherogenic plasma lipid profile and a lower risk of cardiovascular disease than men, but this female advantage disappears after menopause. This suggests that female sex steroids affect lipoprotein metabolism. The impact of variations in the availability of ovarian hormones during the menstrual cycle on lipoprotein metabolism is not known. We therefore investigated whether very-low-density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B-100 (apoB-100) kinetics are different during the follicular (FP) and luteal phases (LP) of the menstrual cycle. We studied seven healthy, premenopausal women (age 27 +/- 2 yr, BMI 25 +/- 2 kg/m(2)) once during FP and once during LP. We measured VLDL-TG, VLDL-apoB-100, and plasma free fatty acid (FFA) kinetics by using stable isotope-labeled tracers, VLDL subclass profile by nuclear magnetic resonance spectroscopy, whole body fat oxidation by indirect calorimetry, and the plasma concentrations of lipoprotein lipase (LPL) and hepatic lipase (HL) by ELISA. VLDL-TG and VLDL-apoB-100 concentrations in plasma, VLDL-TG and VLDL-apoB-100 secretion rates and mean residence times, VLDL subclass distribution, FFA concentration and rate of appearance in plasma, whole body substrate oxidation, and LPL and HL concentrations in plasma were not different during the FP and the LP. We conclude that VLDL-TG and VLDL-apoB-100 metabolism is not affected by menstrual cycle phase.

No effect of menstrual cycle phase on glycerol or palmitate kinetics during 90 min of moderate exercise.

The systemic flux of glycerol and palmitate [a representative nonesterified free fatty acid (NEFA)] was assessed in three different phases of the menstrual cycle at rest and during moderate-intensity exercise. It was hypothesized that circulating glycerol and NEFA turnover would be greatest in the midfollicular (MF) phase of the menstrual cycle, when estrogen is elevated but progesterone low, followed by the midluteal phase (ML; high estrogen and progesterone), and lowest in the early follicular (EF) phase of the menstrual cycle (low estrogen and progesterone). Subjects included moderately active, eumenorrheic, healthy women. Testing occurred after 3 days of diet control and after an overnight fast (12-13 h). Resting and exercise (50% maximal oxygen uptake, 90 min) measurements of tracer-determined glycerol and palmitate kinetics were made. There was a significant increase in both glycerol and palmitate turnover from rest to exercise in all phases of the menstrual cycle (P<0.0001). No significant differences, however, were observed between cycle phases in the systemic flux of glycerol or palmitate, at rest or during exercise. Maximal peripheral lipolysis during exercise, as represented by glycerol rate of appearance at 90 min, equaled 8.45+/-0.96, 8.35+/-1.12, and 7.71+/-0.96 micromol.kg-1.min-1 in the EF, MF, and ML phases, respectively. Circulating free fatty acid utilization also peaked at 90 min of exercise, as indicated by the palmitate rate of disappearance (3.31+/-0.35, 3.17+/-0.39, and 3.47+/-0.26 micromol.kg-1.min-1) in the EF, MF, and ML phases, respectively. In conclusion, systemic rates of glycerol and NEFA turnover (as represented by palmitate flux) were not significantly affected by the cyclic fluctuations in estrogen and progesterone that occur throughout the normal menstrual cycle, either at rest or during 90 min of moderate exercise.

Plasma FFA utilization and fatty acid-binding protein content are diminished in type 2 diabetic muscle.

In this study, we investigated the hypothesis that impairments in forearm skeletal muscle free fatty acid (FFA) metabolism are present in patients with type 2 diabetes both in the overnight fasted state and during beta-adrenergic stimulation. Eight obese subjects with type 2 diabetes and eight nonobese controls (Con) were studied using the forearm balance technique and indirect calorimetry during infusion of the stable isotope tracer [U-(13)C]palmitate after an overnight fast and during infusion of the nonselective beta-agonist isoprenaline (Iso, 20 ng. kg lean body mass(-1) x min(-1)). Additionally, activities of mitochondrial enzymes and of cytoplasmatic fatty acid-binding protein (FABP) were determined in biopsies from the vastus lateralis muscle. Both during fasting and Iso infusion, the tracer balance data showed that forearm muscle FFA uptake (Con vs. type 2: fast 449+/-69 vs. 258 +/-42 and Iso 715+/-129 vs. 398+/-70 nmol. 100 ml tissue(-1) x min(-1), P<0.05) and FFA release were lower in type 2 diabetes compared with Con. Also, the oxidation of plasma FFA by skeletal muscle was blunted during Iso infusion in type 2 diabetes (Con vs. type 2: Iso 446 +/- 274 vs. 16+/-70 nmol. 100 ml tissue(-1) x min(-1), P<0.05). The net forearm glycerol release was increased in type 2 diabetic subjects (P< 0.05), which points to an increased forearm lipolysis. Additionally, skeletal muscle cytoplasmatic FABP content and the activity of muscle oxidative enzymes were lowered in type 2 diabetes. We conclude that the uptake and oxidation of plasma FFA are impaired in the forearm muscles of type 2 diabetic subjects in the overnight fasted state with and without Iso stimulation.

Improved accuracy and precision of gas chromatography/mass spectrometry measurements for metabolic tracers.

The use of stable-isotope tracer methodology to study substrate metabolic kinetics requires accurate measurement of the tracer to tracee ratio (TTR), often by gas chromatography/mass spectrometry (GC/MS). Many approaches for measurement of the TTR by GC/MS do not use standards of known isotopic enrichment to control for variability in instrument response. In addition, most GC/MS applications exhibit some degree of concentration dependency whereby the measured ion abundance ratio varies with the quantity of sample analyzed, thereby placing a limitation on the accuracy of isotopic enrichment standard curves unless the quantities of standards and samples analyzed are closely matched. We document the degree to which day-to-day variability can affect the instrument response for several GC/MS analyses of metabolic tracers when isotopic enrichment standards are not used to control for variable instrument response. Furthermore, we report a new approach that incorporates concentration dependencies within a standard curve to improve the accuracy and precision of TTR measurements over a range of sample quantities analyzed. The new approach was applied to plasma samples obtained from experimental protocols performed in human subjects with three commonly used tracers: 2H2-palmitate, 15N2-urea, and 13C-leucine. Variability in the day-to-day instrument response was 84% and 26% for 2H2-palmitate and 15N2-urea, respectively; in addition, up to 10% variability due to concentration dependency was noted for these applications. The new approach virtually eliminated these sources of variability. After controlling for concentration dependency, a threefold reduction in the standard error was noted when the enrichment of 13C-leucine measured by electron-impact (EI) ionization GC/MS was correlated against negative chemical ionization (NCI) GC/MS. These data demonstrate that our new approach decreases the errors in TTR determination caused by variations in instrument response and concentration dependency. This approach is generically applicable, and can improve the accuracy and precision of TTR determinations for most GC/MS analyses.

Metabolic effects of liver transplantation in cirrhotic patients.

To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.

Fuel choices by human platelets in human plasma.

Despite the fact that homogeneous preparations of isolated cells are now being used very effectively to study a range of important biochemical questions, it is still not known what combination of fuels and energy-producing pathways is used by cells when offered the complex mixture characteristic of plasma or extracellular fluid. We have developed an in vitro system whereby highly purified and functional human platelets are incubated in human plasma that has been minimally modified from its native state. The concentration of platelets and fuels, and the complexity of fuels in the incubation are similar to those in vivo. The preparation thus represents a reasonable approximation of the physiological condition, considering the complex nature of the system being studied. Measurements carried out simultaneously during the incubation are rates of oxygen consumption, lactate production and fuel oxidation. The data allow the calculation of total ATP turnover, and contributions to this turnover by lactate production and the oxidation of individual fuels. Lactate production accounts for 24% of the ATP turnover. The oxidation of glucose and 3-hydroxybutyrate each account for under 5%, palmitate for 21%, oleate for 7% and acetate for 9%, leaving 32% of the ATP turnover as yet unaccounted for. The results confirm some previous measurements in the literature, but show that data collected under non-physiological experimental conditions can be misleading.

Use of gas chromatography/isotope ratio-mass spectrometry to study triglyceride metabolism in humans.

The study of triglyceride (TG) metabolism using stable isotope tracers would be facilitated by being able to detect low 13C enrichment. To meet this goal, we developed a gas chromatography/isotope ratio-mass spectrometry technique to measure the enrichment of palmitate in nonesterified fatty acids (NEFA) and TG as its methyl derivative. This method allows accurate and reproducible measurements of enrichment as low as 0.009 mole percent excess (MPE), in a range between 0-0.65 MPE. The usefulness of this method is shown by two studies of lipid metabolism in human beings. First, we studied the metabolic fate of an oral TG load labeled with [1,1,1-13C3]tripalmitin. Labeled palmitate appeared concurrently in plasma NEFA and TG, and four hours after the load, the labeling was higher in NEFA than in TG (MPE NEFA: 1.53 +/- 0.31 vs. MPE TG: 0.78 +/- 0.06, P < 0.05). In a second study, the hepatic reesterification of NEFA was estimated by measuring the appearance of infused [1-13C]palmitate in circulating TG. The estimated contribution of plasma NEFA to circulating TG increased to a maximum of 22%. Thus, gas chromatography/isotope ratio-mass spectrometry appears to be a useful tool for future studies of lipid metabolism in humans.

Plasma lipoproteins as targeting carriers to tumour tissues after administration of a lipophilic agent to mice.

We synthesized 14C-warfarin hexadecyl ether (14C-WHE) by addition of a palmityl moiety to the hydroxyl group at the 4-position of 14C-warfarin, a compound known to bind to serum albumin. 14C-WHE preferentially bound to the lipoproteins, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), in mouse plasma both in vitro and in vivo. 14C-Warfarin mainly concentrated in the liver immediately after intravenous administration to mice bearing M5076 sarcoma, and was found at only low concentrations in other tissues including the tumour. 14C-WHE highly distributed to the tumour, adrenal, and spleen, as well as the liver. These tissues coincided with those in which human 125I-LDL was vigorously incorporated. The results indicate that chemical modification of an agent, giving it high lipophilicity, will enable it to bind to lipoproteins after intravenous administration. These modifications raise the possibility of lipoproteins as endogenous targeting carriers into tumour cells, which have high LDL-receptor activity.

The sulfur-cyanolysis sites of serum albumin: metabolite competition studies.

In the presence of a source of sulfane sulfur, a cyanolysis reaction catalyzed by serum albumin may contribute to cyanide detoxication. The active site for this catalysis by serum albumin has been investigated in competition studies with ligands that have known albumin binding sites. Despite complications caused by the occurrence of multiple primary and secondary sites for many ligands, the results show that the primary sites for bilirubin, steroids, indoles, aspirin, and palmitate are distinct from that for sulfur. Laurate is a tight-binding partial inhibitor of the cyanolysis reaction, competitive with cyanide rather than with sulfur. In view of the formal mechanism previously established for the catalyzed reaction, this result indicates that the sulfur-cyanolysis site is probably near the site occupied by laurate.

Essential fatty acid deficiency in critically ill surgical patients.

Plasma fatty acid profiles of 33 critically ill surgical patients receiving fat-free parenteral nutrition were examined at weekly intervals up to 28 days. While plasma total fatty acid concentration remained relatively constant and within the normal range, marked compositional alterations were apparent. Levels of linoleate (18:2 omega 6), the major essential fatty acid in man, fell below normal values (754 +/- 259 micrograms/ml) in 67 percent of patients within 1 week after cessation of oral intake. Decreases in other omega 6 unsaturated fatty acids, derived from linoleate, were also apparent. In contrast, gradual increases were observed in levels of endogenously synthesized fatty acids, palmitate (16:0), palmitoleate (16:1) and oleate (18:1 omega 9). A fatty acid unique to essential fatty acid deficiency, 5,8,11 eicosatrienoate (20:3 omega 9), appeared in 25 percent of the patients during the first week and in all patients by the third week of study. Considering the rapid appearance and progression of these biochemical changes, early initiation of linoleate supplementation appears justified to forestall the development of related clinical sequelae.

Biological activity of protein-bound calcium in serum.

We have previously reported the existence of a subfraction of serum calcium that is tightly bound to normal human serum proteins. This tightly bound calcium fraction (TBC) is thought to be a calcium-albumin-fatty acid complex (ACP) because similar complexes can be prepared by the sequential addition of albumin to calcium in the presence of palmitic acid. These studies deal primarily with TBC from rat serum and the uptake of calcium by bone cells in palmitate-treated serum. It is reported that TBC does not exchange with ionized calcium and that calcium binds strongly to albumin in the presence of palmitate. The uptake of calcium in palmitate-treated serum is three times greater than the uptake of calcium in control serum in both in vitro and in vivo systems. These findings demonstrate that 1) a tightly bound albumin-calcium fraction is present in both human and rat sera; 2) calcium in TBC may be complexed to albumin via fatty acids; 3) TBC does not participate in the maintenance of the level of ionized calcium in serum and 4) circulating TBC or ACP complexes may be taken up by living cells.