Intravascular Kinetics Research Articles

Comparison of the Impact of High‐Dose DHA and EPA Supplementation on the Intravascular Kinetics of Very‐Low Density Lipoprotein Apolipoprotein C‐III

BackgroundApolipoprotein C‐III (apoC‐III) is an emerging risk factor for cardiovascular disease (CVD) and its presence on very‐low density lipoproteins (VLDL) may enhance their atherogenicity, primarily by delaying their rate of catabolism. We have shown in a recent study that high‐dose supplementation with docosahexaenoic acid (DHA) led to more pronounced reductions in TG compared with eicosapentaenoic acid (EPA), but mechanisms remain unclear. The objective of this study was to compare the individual effect of EPA and DHA on VLDL‐apoC‐III metabolism in men and women at risk of CVD.MethodsIn a randomized double‐blind crossover trial, 10 men and 8 women with abdominal obesity and low‐grade inflammation were subjected to three 10‐wk supplementation phases: 1) EPA (2.7 g/d); 2) DHA (2.7 g/d); 3) corn oil (0g DHA, 0g EPA, control), separated by a 9‐wk washout. All supplements were provided as 3×1g capsules for a total of 3g/d. After each dietary intervention, subjects received a primed‐constant infusion of [5,5,5‐D(3)]‐L‐leucine for 12h under constant feeding conditions for the determination of VLDL‐apoC‐III kinetics.ResultsBoth DHA (−11.7%, p=0.01) and EPA (−6.4%, p=0.03) similarly reduced the fractional catabolism rate (FCR) of VLDL‐apoC‐III compared with control (p=0.75 between DHA and EPA). On the other hand, neither DHA nor EPA supplementation had a significant impact on VLDL‐apoC‐III concentrations and production rate (PR). Compared with control, DHA‐ and EPA‐induced reductions in TG were correlated with DHA‐ and EPA‐induced variations in VLDL‐apoC‐III PR (r=0.49, p=0.03 and r=0.48, p=0.05 respectively), but not with changes in VLDL‐apoC‐III FCR.ConclusionsHigh‐dose supplementation with DHA or EPA is associated with reduced clearance of VLDL‐apoC‐III, with no effect, however, on VLDL‐apoC‐III concentrations and production. Although the DHA‐ and EPA‐induced reductions in serum TG may be more sensitive to treatment‐induced variations in VLDL‐apoC‐III production, other mechanisms must be investigated to explain why DHA may reduce serum TG more than EPA.Support or Funding InformationFinancial support for this RCT was provided exclusively by a grant from the Canadian Institutes for Health Research (MOP‐123494). Douglas Laboratories provided the EPA, DHA and placebo capsules used in this study. The funding source was not involved in the study design, conduct of the study, or collection, management, analysis, or interpretation of the data or in the preparation or review of the manuscript and had no right to approve or disapprove of the submitted manuscript. CV is a fellow of the French Foundation for Medical Research (FRM, file code: 40303). JA is a recipient of a PhD Scholarships from the Canadian Institutes for Health Research and the Fonds de recherche du Québec – Santé.

N-3 Polyunsaturated Fatty Acid Supplementation Has No Effect on Postprandial Triglyceride-Rich Lipoprotein Kinetics in Men with Type 2 Diabetes.

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been proposed to modulate plasma lipids, lipoprotein metabolism, and inflammatory state and to reduce triglyceride (TG) concentrations. The present double-blind, randomized, placebo-controlled, crossover study investigated the effects of n-3 PUFA supplementation at 3 g/d for 8 weeks on the intravascular kinetics of intestinally derived apolipoprotein (apo) B-48-containing lipoproteins in 10 men with type 2 diabetes. In vivo kinetics of the TG-rich lipoprotein (TRL) apoB-48 and VLDL apoB-100 were assessed using a primed-constant infusion of L-[5,5,5-D3] leucine for 12 hours in a fed state. Compared with the placebo, n-3 PUFA supplementation significantly reduced fasting TG concentrations by −9.7% (P = 0.05) but also significantly increased plasma levels of cholesterol (C) (+6.0%, P = 0.05), LDL-C (+12.2%, P = 0.04), and HDL-C (+8.4, P = 0.007). n-3 PUFA supplementation had no significant impact on postprandial TRL apoB-48 and VLDL apoB-100 levels or on the production or catabolic rates of these lipoproteins. These data indicate that 8-week supplementation with n-3 PUFAs in men with type 2 diabetes has no beneficial effect on TRL apoB-48 and VLDL apoB-100 levels or kinetics.

Abstract 167: Dietary Medium-chain Triglyceride Supplementation Has No Effect on Apolipoprotein B-48 and Apolipoprotein B-100 Kinetics in Insulin-resistant Males

Medium-chain triglyceride (MCT) supplements are used by clinicians to treat patients with severe hypertriglyceridemia at risk for pancreatitis. However, the potential mechanisms underlying the triglyceride-lowering effects of MCT have not been thoroughly examined yet in human. This double-blind crossover study compared the impact of a 4-week supplementation with MCT oil 20 g/d vs corn oil 20 g/d on the intravascular kinetics of apolipoprotein (apo) B-48-containing triglyceride-rich lipoproteins (TRL) and apoB-100-containing VLDL as well as on the expression of key intestinal genes involved in lipid metabolism in 28 obese insulin-resistant males. In vivo kinetics of TRL apoB-48 and VLDL apoB-100 were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12h in the fed state. Expression of key intestinal genes involved in lipid metabolism was assessed in a subgroup of participants (n=16) by real-time PCR quantification in duodenal biopsy performed at the end of each phase of the supplementation in the fasted state. Compared with corn oil, MCT supplement had no significant effect on plasma lipoprotein profile and the TRL apoB-48 and VLDL apoB-100 kinetics. There were significant positive correlations at the end of the MCT supplementation between expression of SREBP-2 and expression of HNF-4α (r=0.51; P<0.05), HMGCoAR (r=0.75; P<0.001), ACAT-2 (r=0.51; P<0.05), NPC1L1 (r=0.66; P<0.05), ACS-1 (r=0.74; P<0.001), LDL receptor (r=0.67; P<0.05) and PCSK-9 (r=0.60; P<0.05) in intestinal tissue. However, MCT supplement had no significant impact on the expression of those key lipid and fatty acid regulation genes compared with corn oil. These data indicate that 4-week supplementation with MCT in obese insulin resistant men has no effect on TRL apoB-48 and VLDL apoB-100 kinetics and on the intestinal expression of genes involved in lipid and fatty acid metabolism.

Adding MUFA to a dietary portfolio of cholesterol-lowering foods reduces apoAI fractional catabolic rate in subjects with dyslipidaemia

The present randomised parallel study assessed the impact of adding MUFA to a dietary portfolio of cholesterol-lowering foods on the intravascular kinetics of apoAI- and apoB-containing lipoproteins in subjects with dyslipidaemia. A sample of sixteen men and postmenopausal women consumed a run-in stabilisation diet for 4 weeks. Subjects were then randomly assigned to an experimental dietary portfolio either high or low in MUFA for another 4 weeks. MUFA substituted 13·0% of total energy from carbohydrate (CHO) in the high-MUFA dietary portfolio. Lipoprotein kinetics were assessed after the run-in and portfolio diets using a primed, constant infusion of [2H3]leucine and multicompartmental modelling. The high-MUFA dietary portfolio resulted in higher apoAI pool size (PS) compared with the low-MUFA dietary portfolio (15·9% between-diet difference, P¼0·03). This difference appeared to be mainly attributable to a reduction in apoAI fractional catabolic rate (FCR) after the high-MUFA diet (25·6%, P¼0·02 v. pre-diet values), with no significant change in production rate. The high-MUFA dietary portfolio tended to reduce LDL apoB100 PS compared with the low-MUFA dietary portfolio (228·5% between-diet that adding MUFA to a dietary portfolio of cholesterol-lowering foods provides the added advantage of raising HDL primarily through a reduction in HDL clearance rate. Replacing CHO with MUFA in a dietary portfolio may also lead to reductions in LDL apoB100 concentrations primarily by increasing LDL clearance rate, thus potentiating further the well-known cholesterol-lowering effect of this diet.

Use of 111-Indium–labeled autologous eosinophils to establish the in vivo kinetics of human eosinophils in healthy subjects

AbstractEosinophils are the major cellular effectors of allergic inflammation and represent an important therapeutic target. Although the genesis and activation of eosinophils have been extensively explored, little is known about their intravascular kinetics or physiological fate. This study was designed to determine the intravascular life span of eosinophils, their partitioning between circulating and marginated pools, and sites of disposal in healthy persons. Using autologous, minimally manipulated 111-Indium–labeled leukocytes with blood sampling, we measured the eosinophil intravascular residence time as 25.2 hours (compared with 10.3 hours for neutrophils) and demonstrated a substantial marginated eosinophil pool. γ camera imaging studies using purified eosinophils demonstrated initial retention in the lungs, with early redistribution to the liver and spleen, and evidence of recirculation from a hepatic pool. This work provides the first in vivo measurements of eosinophil kinetics in healthy volunteers and shows that 111-Indium–labeled eosinophils can be used to monitor the fate of eosinophils noninvasively.

T3 Measuring eosinophil kinetics in humanS

<h3>Introduction and Objectives</h3> Eosinophils are major cellular effectors of allergic inflammation and represent an important therapeutic target in asthma. While much is understood about the generation and activation of eosinophils, little is known about their intravascular kinetics and physiological fate. The purpose of this study was to image sites of eosinophil distribution and measure eosinophil kinetics in healthy individuals using autologous 111-Indium-labelled eosinophils. <h3>Methods</h3> To determine “gold standard” kinetics of minimally-manipulated eosinophils<b>,</b> mixed leucocytes were isolated from the blood of healthy volunteers, labelled with 111-Indium-tropolonate and re-injected. Blood was sampled 0.75–72 h post-injection. Neutrophils and eosinophils were isolated in parallel, and cell-associated radioactivity was measured. To image sites of eosinophil margination/uptake eosinophils were purified using plasma-Percoll gradients and anti-CD16 immunomagnetic beads, labelled with 111-Indium-tropolonate and re-injected. The distribution of eosinophils was recorded by γ camera dynamic imaging (0–40 min) followed by static imaging up to 72 h. <h3>Results</h3> Using minimally manipulated granulocytes we found that the 45min neutrophil recovery was 57±10% (n=7) and the intravascular lifespan was 10.3±0.1 h, in agreement with previous studies. By contrast, the 45min recovery of eosinophils was 15±2% (n=7) and eosinophil lifespan was 25.2±3.8 h. Moreover, eosinophils appeared to re-circulate at ∼4 h and 9 h before monoexponential removal. Using 111-Indium-eosinophils and γ camera imaging, we demonstrated initial retention of cells in the lungs, clearing to baseline by 40 min, with some early accumulation in the liver and progressive accumulation in the spleen (n=3). Simultaneous blood sampling showed that the 45 min recovery and intravascular lifespan of purified labelled eosinophils were 11±2% and 29.3±2.1 h, respectively, comparable to minimally manipulated cells. Purified cells also exhibited recirculation at ∼6 h and 12 h. Of note, the disappearance of eosinophils from the liver at 6 h and 9 h coincided with their re-appearance in circulating blood, suggesting the liver as a possible site of transient eosinophil sequestration. <h3>Conclusions</h3> This work provides the first in vivo measurements of eosinophil kinetics in healthy volunteers. Our data suggest that 111-In-labelled-eosinophils can be used to monitor the organ distribution and fate of eosinophils non-invasively. This technique may have an important role in assessing the therapeutic effects of eosinophil-targeted drugs.

Intravascular kinetics of C-reactive protein and their relationships with features of the metabolic syndrome

Intravascular kinetics of C-reactive protein and their relationships with features of the metabolic syndrome

Intravascular Kinetics of C-Reactive Protein and Their Relationships with Features of the Metabolic Syndrome

The objective of the study was to describe, for the first time, the intravascular kinetics of C-reactive protein (CRP), using stable isotopes, and its relationship with features of the metabolic syndrome. Sixteen men and 16 women [aged 49 +/- 9 years, body mass index (BMI) 28.7 +/- 4.5 kg/m(2)] underwent a 12-h primed-constant infusion of 5,5,5-(2)H(3)-l-leucine. CRP was purified from the plasma fraction rho greater than 1.25 g/ml by affinity chromatography, and isotopic enrichment over time was determined by gas chromatography/mass spectrometry. The CRP fractional catabolic rate was 60% higher in men than women (0.49 +/- 1.83 vs. 0.30 +/- 1.80 pool/d, P = 0.03), but this difference was no longer significant in a multivariate model that included several features associated with the metabolic syndrome. The CRP production rate (PR) and pool size were not statistically different between sexes. Plasma CRP concentrations were more strongly correlated with the PR (r = 0.80, P < 0.0001) than with the fractional catabolic rate of CRP (r = 0.39, P < 0.05). The PR of CRP was positively correlated with waist girth (r = 0.53, P < 0.01), plasma low-density lipoprotein apolipoprotein B-100 (r = 0.42, P = 0.07), triglyceride (r = 0.41, P = 0.06), and IL-6 concentrations (r = 0.61, P = 0.0008) and inversely correlated with high-density lipoprotein-cholesterol (r = -0.47, P = 0.03) and adiponectin (r = -0.63, P < 0.0005) after adjustment for sex. Blood pressure and low-density lipoprotein-cholesterol showed no association with CRP kinetics. The PR of CRP appeared as the main determinant of CRP concentrations and showed significant associations with features of the metabolic syndrome as well as with adipose tissue-derived cytokines such as IL-6 and adiponectin.

Recombinant Human Thyroid-Stimulating Hormone

The major functions of pituitary thyroid-stimulating hormone (TSH) are to maintain the biosynthesis and secretion of the thyroid hormones L-thyroxine (T4) and L-3,5,3'triidothyronine (T3). The TSH core contains two apoproteins, the alpha and beta subunits. The alpha subunit is identical to that of pituitary follitropin, pituitary lutropin and placental chorionic gonadotropin, whereas the beta subunit is unique. TSH is a glycoprotein; the glycoprotein components of the alpha and beta subunits account for more than 10% of their mass and are essential for normal thyrotropic action and intravascular kinetics. The hypothalamic tripeptide, TSH-releasing hormone (TRH) is required for optimum TSH biosynthesis, particularly as far as addition of the glycoprotein components is concerned. TRH deficiency is associated with secretion of TSH molecules that are appropriately measured in most assays but have reduced bioactivity. In previous years the TSH used in clinical practice was obtained and purified from bovine pituitaries. Bovine TSH was used to test thyroid function and to augment the uptake of radioiodine in patients with thyroid cancer. Bovine TSH has been largely abandoned as a clinical agent because of adverse immune reactions. A recombinant human TSH (rhTSH; Thyrogen), has been approved by the US FDA for diagnostic use in patients with thyroid cancer. The alpha and beta subunits of Thyrogen are identical to those of human pituitary TSH. Thyrogen has a specific activity of approximately 4 IU/mg and is a potent stimulator of T4, T3 and thyroglobulin (Tg) secretion in healthy volunteers. It also increases thyroid iodide uptake in patients with thyroid cancer or multinodular goitre and in volunteers, even those exposed to large amounts of stable iodide. Thyroid cancer patients who have been treated by thyroidectomy and radioiodine ablation but are at risk of harbouring residual thyroid cancer are candidates for Thyrogen administration to prepare them for whole body iodide scans and serum Tg measurements. In thyroidectomised thyroid cancer patients who are unable to secrete pituitary TSH upon thyroid hormone withdrawal, Thyrogen is the only acceptable method to prepare them for these procedures. Thyrogen has been used on a compassionate basis to prepare patients for radioiodine ablation. rhTSH, in addition to being useful in the management of patients with thyroid cancer, is potentially useful to test thyroid reserve and to aid in thyroid-related nuclear medicine procedures. In the future, TSH analogues that have superagonist or antagonist properties may become available as therapeutic agents.

Effect of intravascular ligand binding on parameter estimates derived from tracer kinetic modelling

The purpose of this study was to assess the effect of intravascular ligand binding on parameter estimates derived from tracer kinetic modelling. To this end intravascular ligand kinetics between the free and a bound compartment in plasma and the exchange of tracer between the capillary space and tissue were analysed using a simple compartment model. The effect of non-equilibrated intravascular compartments on parameter estimates was evaluated in a computer simulation. It was found that three kinetic situations must be distinguished. If the intravascular compartments are fully equilibrated when the ligand reaches the target organ, intravascular binding simply acts as a scale factor for the transport-related parameter K1. If the intravascular kinetics is very slow, only minimal binding will occur. In between there is a range where ongoing equilibration leads to time variability of K1. Since tracer kinetic modelling usually does not account for such time variability, the parameter estimates become biased, the degree of the bias depending on the intravascular binding kinetics. Furthermore the bias may be dependent on receptor density, meaning that model-derived receptor estimates are not linearly related to the true receptor density. It is concluded that intravascular ligand binding can severely affect parameter estimates derived from tracer kinetic modelling. Especially disturbing are effects due to ongoing intravascular equilibration following the arrival of the ligand in the target organ. These can be avoided by letting the ligand equilibrate with blood in a syringe prior to injection.

Effects of storage of granulocytes on their fate in vivo.

During storage of granulocytes, the chemotactic response deteriorates first and to the greatest extent. Because in vitro assays such as chemataxis may not adequately predict the fate of granulocytes in vivo, we determined the intravascular kinetics and migration into skin windows of indium-111 labeled granulocytes. Granulocytes stored for 24 hours at either 1 to 6 or 20 to 24 degrees C had significantly reduced intravascular recoveries and migration into skin windows. Granulocytes stored for only eight hours at 20 to 24 degrees C had normal intravascular recovery and migration into skin windows. In granulocytes stored for eight hours at 1 to 6 degrees C, the intravascular recovery and migration into skin windows were reduced. Because of the method of calculating migration, this observation suggests that granulocytes which do circulate after storage at 1 to 6 degrees C had decreased ability to migrate into the skin windows. Granulocytes can be stored for up to eight hours at 20 to 24 degrees C without reduction in their ability to circulate and migrate to a site of inflammation. However, since many practical issues in granulocyte storage are not resolved, granulocytes should be transfused as soon as possible after collection.

Comparison Study of the Kinetics of Ceftizoxime Penetration into Extravascular Spaces with Known Surface Area/Volume Ratio In Vitro and In Vivo in Rabbits

The extravascular kinetics of ceftizoxime were studied both in an in vitro kinetic model and in an in vivo rabbit model. Visking tubing chambers were used in both models to provide extravascular spaces with large or small volumes and surface areas, but identical surface area/volume ratios. Four rabbits, each implanted with two large Visking chambers and four small chambers, received 50 mg of ceftizoxime per kg intramuscularly every 3 h for eight doses. In the in vitro model, 80 mg of ceftizoxime was infused over 30 min every 3 h for eight doses. Intravascular and extravascular spaces were sampled in both models after the eighth dose. Ceftizoxime had similar intravascular kinetics in both models, i.e., the peak levels, the peak-to-trough fluctuations, and the half-life were comparable. The area under the curve (AUC) for the extravascular spaces was also similar in the two models. Large and small chambers having identical surface area/volume ratios demonstrated identical kinetics. The extravascular Visking chamber spaces achieved equilibrium with the intravascular spaces in both models, i.e., the AUC for the extravascular spaces was the same (P > 0.2) as that for the serum (rabbit model) or the test chamber (in vitro model). This study illustrates (i) that our modified in vitro model is a potentially valid model for studying extravascular kinetics; (ii) that extravascular spaces with identical surface area/volume ratios show similar penetration kinetics with a freely diffusible drug, such as ceftizoxime, despite differences in size; and (iii) that the Visking chamber extravascular-space model permits the free diffusion of the antimicrobial agent and reaches equilibrium (equivalent AUC) with the intravascular space.

In Vitro Function and Post-Transfusion Survival of Granulocytes Collected by Continuous-Flow Centrifugation and by Filtration Leukapheresis

The function of granulocytes collected by continuous-flow centrifugation (CFC) and by filtration leukapheresis (FL) was studied in vitro, and the post-transfusion recovery and intravascular survival of these cells was studied by autologous transfusion in normal donors. Granulocytes collected by both FL and CFC leukapheresis (CFCL) functioned normally in the quantitative nitroblue tetrazolium, oxygen consumption, and chemotaxis assays. Bacterial killing was slightly but consistently decreased in FL but not CFCL granulocytes. The post-transfusion recovery of control granulocytes collected by ordinary phlebotomy averaged 52% in eight transfusions, compared with 34% for six CFCL granulocyte concentrates and 16% for six FL concentrates. The intravascular half-times were 3.8 hr for phlebotomy and 3.0 hr for CFCL granulocytes. FL granulocytes had survival curves which were nonlinear and a single half-life could not be calculated. The average half-time 30 min after transfusion was 1.3 hr, and 3 hr after transfusion it was 2.6 hr. Granulocytes collected by FL had a mild impairment of bacterial killing, decreased post-transfusion recovery, and altered intravascular kinetics. None of these abnormalities was found in granulocytes collected by CFCL.

In vitro function and post-transfusion survival of granulocytes collected by continuous-flow centrifugation and by filtration leukapheresis

The function of granulocytes collected by continuous-flow centrifugation (CFC) and by filtration leukapheresis (FL) was studied in vitro, and the post-transfusion recovery and intravascular survival of these cells was studied by autologous transfusion in normal donors. Granulocytes collected by both FL and CFC leukapheresis (CFCL) functioned normally in the quantitative nitroblue tetrazolium, oxygen consumption, and chemotaxis assays. Bacterial killing was slightly but consistently decreased in FL but not CFCL granulocytes. The post- transfusion recovery of control granulocytes collected by ordinary phlebotomy averaged 52% in eight transfusions, compared with 34% for six CFCL granulocyte concentrates and 16% for six FL concentrates. The intravascular half-times were 3.8 hr for phlebotomy and 3.0 hr for CFCL granulocytes. FL granulocytes had survival curves which were nonlinear and a single half-life could not be calculated. The average half-time 30 min after transfusion was 1.3 hr, and 3 hr after transfusion it was 2.6 hr. Granulocytes collected by FL had a mild impairment of bacterial killing, decreased post-transfusion recovery, and altered intravascular kinetics. None of these abnormalities was found in granulocytes collected by CFCL.

Chromate transport in human leukocytes

Chromium is a trace metal of importance in human physiology and, in addition, as 51-chromate, has been extensively used as a label in the study of blood cell pool sizes and intravascular kinetics. The transport characteristics of 51-chromate were investigated in normal human leukocytes. Chromate uptake is unidirectional over a 1 hr incubation with extracellular chromate concentrations up to 200 mumoles/liter. Under these conditions, intracellular 51-chromium is in a form in which it is nonexchangeable. Influx is temperature sensitive with a Q(10) of approximately 2 and may be energy dependent since a variety of metabolic poisons strongly inhibit uptake. The unidirectional influx of chromate follows Michaelis-Menten kinetics; the maximum velocity is 52 mmumoles/g dry weight of cells per min and the chromate concentration at which influx velocity is half maximal is 87 mumoles/liter. This transport mechanism is highly specific for chromate; other divalent tetrahedral anions only slightly inhibit influx at concentrations up to 10 times that of chromate. Metavanadate, however, competitively inhibits chromate influx at equimolar concentrations. Exposure of cells to unlabeled chromate leads to inhibition of subsequent influx of 51-chromate. It is suggested that this is due to a primary inhibitory effect of chromate on cellular energy metabolism.