Characterization of chylomicron remnant clearance by retinyl palmitate label in normal humans.

To characterize chylomicron remnant clearance by the liver, plasma elimination of retinyl palmitate-labeled chylomicron remnants was studied in 18 healthy subjects, ages 21-42 years. Autologous plasma containing retinyl palmitate-labeled chylomicrons and their remnants was injected intravenously, and retinyl palmitate disappearance was measured in serial plasma samples in all subjects and in lipoprotein fractions in 11 subjects. The injected doses (n = 18) ranged from 0.34 to 7.11 mumol retinyl palmitate in d less than or equal to 1.006 g/ml particles with an average molar ratio of 330/1 of retinyl palmitate/apoB-48 (n = 8). The label distributed in the intravascular space and exhibited apparent first order elimination, monoexponential in 6 and biexponential in 12 subjects. The first rapid component k1 (t1/2 18.8 +/- 11.4 min, n = 18) was shown to represent retinyl palmitate in particles of d less than or equal to 1.006 g/ml, i.e., chylomicron remnants, and the second slow component k2 (t1/2 123 +/- 62 min, n = 12) small amounts of retinyl palmitate (11 +/- 7%) injected in d greater than 1.006 g/ml particles (therefore excluded from analysis). Assuming a single-compartment model, initial rates of elimination (= dose x k1) of labeled chylomicron remnants obeyed (P = 0.06) Michaelis-Menten saturation kinetics: Km was 921 +/- 305 nmol retinyl palmitate label and Vmax 124 +/- 14 nmol/min corresponding to 0.88 nM apoB-48 for Km and 0.25 x 10(-3) nmol apoB-48.min-1.g-1 liver for Vmax. Their elimination was limited neither by the injected triglyceride dose nor theoretically by the liver blood flow. After the intake of 70 g of fat (cream) containing retinyl palmitate, the plasma retinyl palmitate concentration exceeded the estimated saturation concentration for 7 h. In conclusion, physiological chylomicron remnant catabolism by the liver appears to be saturable by ordinary lipid intake in healthy humans.     

Decreased hepatic retinyl palmitate hydrolase activity in protein-deficient rats

28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmol per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically 'active' retinoids available for normal physiological functions.     

Incorporation of labeled cholesterol esters into chylomicrons in vitro

Labeled cholesterol esters were incorporated into chylomicrons in vitro by adding an acetone solution of the cholesterol ester to a suspension of washed chylomicrons. The physical and metabolic properties of the added labeled ester were the same as those of cholesterol esters incorporated into chylomicrons in vivo, except for the added ester being differently distributed in chylomicrons of different size.     

Hydrolysis of cis and trans isomers of retinyl palmitate by retinyl ester hydrolase of pig liver

Relative retinyl ester hydrolase activities of pig liver homogenates (n = 4) toward 9,13-cis-, 13-cis-, 9-cis-, and all-trans-retinyl palmitate were 6.8 +/- 0.5 (SE), 5.7 +/- 0.5, 2.4 +/- 0.1, and 1, respectively. The range of apparent Km values for the four isomers was 142 to 268 microM, and the pH optima were 8-9 in all cases. Peak activities of retinyl ester hydrolase activities in pig liver cytosol toward 13-cis- and all-trans-retinyl palmitate were found in the 20 to 40% and in the 60 to 80% saturated ammonium sulfate (AS) fractions, respectively. By use of size-exclusion chromatography in 2 M KCl, hydrolase activity eluted at volumes corresponding to greater than 2000, 180, and 15 kDa from the 20-40% AS fraction, and at 180 kDa from the 60-80% AS fraction. On the basis of molecular size, different substrate specificities, detergent effects, and susceptibilities to inhibition by phenylmethylsulfonyl fluoride, we conclude that at least three distinct retinyl ester hydrolases are present in pig liver cytosol.     

Spontaneous transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles

The transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles was studied by resonance energy transfer. The retinoic acid transfers spontaneously between single unilamellar vesicles with a first order rate constant of 9.6 s-1 at 15 degrees C and pH 7.4. At 30 degrees C, the transfer rate was 3.5 times faster than that at 10 degrees C. At pH 7.4, the transfer rate was almost 2 orders of magnitude faster than that observed at pH 1.6. Increasing the concentration of NaCl decreased the retinoic acid transfer rate significantly. Retinyl acetate transfers with a rate constant of 0.15 s-1, but no spontaneous transfer of retinyl palmitate was observed over 60 min. The evidence supports the proposal that retinoic acid and retinyl acetate transfer between single unilamellar vesicles occur via the aqueous phase. In contrast, no spontaneous transfer of retinyl palmitate was observed. However, linear free energy relationships and the thermodynamic parameters for retinyl acetate transfer permit the calculation of rate constant for retinyl palmitate transfer.     

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Plasma sialyltransferase activity in healthy subjects and atherosclerotic patients

Plasma sialyltransferase activity measured by incorporation of cytidine 5;-phospho[14C]acetylneuraminic acid (CMP-NeuAc) into asialofetuin was twofold higher in patients with documented atherosclerosis than in healthy donors. Kinetic studies showed that the enzyme affinity for CMP-NeuAc is the same in donors and patients. Low activity of plasma sialyltransferase in donors may be due to low blood content of this enzyme.     

Plasma adrenomedullin response to maximal exercise in healthy subjects.

The aim of the study was to find out whether maximal exercise performed by healthy young men influences plasma adrenomedullin concentration (ADM) and is the peptide level related to the cardiovascular, metabolic and hormonal changes induced by exercise. Ten subjects (age 24+/-1.0 yr) participated in the study. They performed graded bicycle ergometer exercise until exhaustion. Heart rate (HR) and blood pressure (BP) were measured throughout the test. Before and at the end of exercise venous blood samples were taken for [ADM], noradrenaline [NA], adrenaline [A], growth hormone [hGH], cortisol and lactate [LA] determination. Plasma [ADM] decreased during exercise from 1.71+/-0.09 to 1.53+/-0.10 pmol x l(-1) (p<0.01). This was accompanied by increases in plasma catecholamines and [hGH], while plasma cortisol level did not change. Positive correlation was found between the exercise-induced decreases in plasma ADM and diastolic BP. Blood [LA], systolic and mean BP at the end of exercise correlated negatively with plasma [ADM]. No significant interrelationships were found between plasma ADM, catecholamines or the other hormones measured. The present data suggests, that maximal exercise inhibits ADM secretion in young healthy men. Metabolic acidosis and a decrease in peripheral resistance might be involved in this effect.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

Gait analysis parameters of healthy human subjects with asymmetric loads

This article focuses on the analysis of gait parameters, ground reaction forces (GRF), and motion signals, for the various asymmetric loads carried by healthy human subjects during walking. Eight asymptomatic human volunteers were enrolled in this study. They were asked to walk, at self-selected pace, with various weights ranging from 0 to 11.33 kg (25 lbs) in 2.26 kg (5 lbs) increments, in one hand on a wooden area equipped with a force platform. Moreover, motion data were recorded from lumbar L1 vertebrae at a frequency of 120 Hz. Three trials of data have been recorded for each subject. In order to quantify the effect of increasing loads on the GRF we define the compression area, restitution area, and coefficient of restitution (COR) for GRF curves. We observe an increase in the compression area with respect to the load and almost constant values for the COR. For motion signals analysis we employ wavelet theory. The signals obtained from the lumbar L1 sensor of the spine vertebrae show a decrease in the wavelet detail energy, for the levels 3, 4, and 5, with respect to increasing loads.     

Lipoprotein lipase expression level influences tissue clearance of chylomicron retinyl ester

Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis.In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.