Phospholipids bound with plasma proteins in children with vitamin D-deficient rickets

Composition of phospholipids bound with plasma proteins in the healthy children and in those with vitamin-D-deficient rickets are studied. It is found that the quantitative and qualitative content of phospholipids in the low- and high-density lipoproteins increases considerably with the rickets. At the same time the content of phospholipids which form complexes wit fibrinogen gets two times lower. The character of changes in the phospholipid composition in protein fractions and in the whole plasma in case of rickets is different.     

Genetic aspects of vitamin D-deficient rickets: genetic markers of blood]

Polymorphism of the AB0 blood groups, haptoglobin Hp, vitamin-D-binding protein (Gc), transferrin (Tf), alpha 1-antitrypsin (alpha 1-AT) and serum alkaline phosphatase (Pp) was studied in a group of children suffering from rickets (VDDR) and in a adequate control group of healthy individuals of the same sex-age composition. Considerable differences were revealed between the VDDR patients and healthy individuals in frequencies of the PIM1 and PIM2 factors on the alpha 1-AT system, r and p of the AB0 system as well as the Hp. Increase in a portion of one of the homozygotes for the Hp and for the alpha 1-AT system took place at the expense of other homozygote proportion (the latter being decreased). Heterozygotes frequencies remained intact in both compared groups. Atypical combination of phenotypes and gene frequencies was observed in a group of patients in the alpha 1-AT and AB0 systems as compared with usual distribution in European population. Higher frequencies of rare alleles of the loci under study were observed in the VDDR patients, which is partially reflected in increase in heterozygosity level in total within a cogort of patients analysed. Combination of the Hp 1-1 (Hp)--A(AB0)--M2M2 (alpha 1-AT) factors should be considered as unfavourable in rickets prognosis.     

Pain threshold and morphine activity in vitamin D-deficient rats

In rats, vitamin D-deficiency increases basal pain threshold and the analgesic effect of morphine (hot plate test). Cholecalciferol (1000 I.U./Kg/day s.c.x 5 days) restores pain sensitivity in vitamin D-deficient rats and brings the analgesic effect of morphine back to normal. On the other hand, tolerance to morphine develops faster in vitamin D-deficient rats, this effect too being prevented by cholecalciferol treatment. These data suggest a role for vitamin D status in pain sensitivity and opiate activity.     

Structural-functional changes in the cardiovascular system during high athletic activity in aboriginals of Yakutia

To study structural-functional changes in the cardiovascular system and metabolic characteristics in residents of Yakutia who were engaged in sports activities, 146 Yakut men 18–29 years old living in Yakutsk were examined. The basic group consisted of elite athletes (free-style wrestling and boxing) (n = 108; average age, 20.2 ± 0.2 years). The control group consisted of untrained healthy men (n = 38; average age, 24.5 ± 0.4 years). The examination included primary inspection, anthropometry, echocardiography, integral rheography of the body, measurement of blood pressure (BP), and biochemical analysis of blood serum. The basic group was divided into two subgroups depending on the BP level (Ia, with optimal and normal BP; Ib, with high normal BP).     

Lipoxygenases of animals: changes in lipoxygenase activity from reticulocytes during interaction with blood plasma lipoproteins

A homogeneous sample of lipoxygenase from rabbit reticulocyte cytosol was obtained. The rate of exogenous arachidonate oxidation by reticulocyte lipoxygenase was found to increase in the presence of various blood plasma lipoproteins of mammals. The rate of arachidonate oxidation by soybean lipoxygenase in the presence of these lipoproteins was either unaffected or decreased. The lipoproteins immobilized the substrate; however, preliminary "saturation" of the lipoproteins by a manyfold excess of non-oxidizable oleate did not eliminate the enzyme activation by the lipoproteins. It was concluded that lipoxygenase activation is due to conformational changes of the enzyme during its interaction with membrane components.     

Intra-individual variation of plasma lipids and lipoproteins in prepubescent children

Estimates of the average intra-individual biological variability for plasma lipids and lipoproteins differs substantially among published studies. Moreover, this topic does not appear to have received consideration in exercise and health literature with normal, healthy children as subjects. The purpose of this study was, therefore, to determine the short-term, day-to-day variability of the lipid-lipoprotein profile from 19 children [mean (SD), 11.5 (0.8) years] from 3 separate venous blood samples. Intra-individual standard deviations, variances and coefficients of variance were determined for total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein-cholesterol (HDL-C), HDL-C sub-fractions HDL2 and HDL3, and low-density lipoprotein-cholesterol (LDL-C). The intra-individual variation for TC and LDL-C was used to calculate the 95% confidence intervals (CIs) around the National Cholesterol Education Programme (NCEP 1991) cut-off points. The main finding was that all of the measured blood analytes including TC, TG, HDL-C, HDL2, HDL3, and LDL-C varied considerably from day-to-day. Coefficients of total variation ranged from 3.5% for HDL3 to 25.4% for TG. Classification of individuals using NCEP guidelines was difficult based on only one or two blood samples. The magnitude of variation for LDL-C meant that a 95% CI could not be constructed around the NCEP borderline-high classification from either one or two samples. However, averaging three TC and LDL-C measurements increased the likelihood of classification within the 95% CI. The results indicate that when using the NCEP guidelines for children and adolescents, true concentrations for TC and LDL-C should be based on the mean of multiple samples.     

Calcium and vitamin D metabolism in hypocalcemic vitamin D-resistant rickets carriers

BACKGROUND/AIMS: Hypocalcemic vitamin D-resistant rickets (HVDRR) is a rare monogenic autosomal recessive disorder associated with mutations in the gene of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D3 action. Although many investigations have discussed the clinical manifestations and molecular etiology of this disease, only a few have investigated the biochemical and hormonal status of heterozygous HVDRR. The aim of the current work was to investigate the profile of selected biochemical and hormonal parameters related to the vitamin D endocrine system in a large number of HVDRR heterozygotes. METHODS: 67 relatives of 2 HVDRR patients, all members of an extended Greek kindred of five generations with a common ancestor, were included in the study. Direct sequencing was used to identify VDR gene mutations. Serum Ca, P, 25(OH)D, iPTH, and 1,25(OH)2D levels were determined in all members of the kindred. RESULTS: DNA analysis of the participants led to the design of two study groups: the HVDRR carriers (24) and the control subjects (43). Our results showed elevated circulating serum levels of 1,25(OH)2D3 and lower levels of PTH than their age- and sex-matched controls. No hypocalcemia or hypophosphatemia were detected in HVDRR carriers. CONCLUSIONS: Our findings suggest that HVDRR carriers may have compensatory elevated serum levels of 1,25(OH)2D3 through which they restrain PTH secretion. The study of HVDRR carriers could be a useful tool for the investigation of the vitamin D endocrine system.     

Characterization of dietary phosphorus-dependent duodenal calcium uptake in vitamin D-deficient chicks

The effect of dietary phosphorus on intestinal calcium uptake was examined in duodenal cells isolated from vitamin D-deficient chicks. Cells from chicks on a high phosphorus diet accumulated calcium at a rate 38% higher than cells from animals on a normal phosphorus diet. Diet high in calcium did not affect calcium absorption in duodenal cells. The dietary phosphorus effect on calcium absorption was specific. Uptake of -methyl glucoside was not altered. Increase in calcium absorption by a high phosphorus diet was not due to a change in cellular energy metabolism nor to the content of phosphorus in cells. Kinetically, a high phosphorus diet increased the V _max of calcium uptake; the affinity for calcium was unaffected. The effectiveness of dietary phosphorus to enhance the intestinal calcium uptake could also be demonstrated in brush border membrane vesicles. The increase in calcium uptake was not due to an alteration in membrane binding capacity nor to calcium efflux from vesicles. To test the hypothesis that a high phosphorus diet may affect membrane transport by altering phospholipid metabolism in duodenal cells, we examined the phospholipid content in isolated brush border membranes. The content of phosphatidylcholine, phosphatidylserine, phosphatidyinositol and phosphatidylethanolamine was not altered by the high phosphorus diet. These findings suggest that the vitamin D-independent and dietary phosphorus-dependent effect on intestinal calcium absorption was primarily due to a change in the calcium flux at the luminal side of the cells. However, the precise mechanism is still not clear.     

Beta-blockade with intrinsic sympathetic activity modifies favorably exercise-induced changes in plasma lipoproteins

The effects of beta-blockade on acute exercise-induced changes in plasma lipoprotein levels were investigated in 12 healthy normotensive subjects by use of beta-blockers of three types: a nonselective agent, a beta 1-selective agent, and a nonselective agent with intrinsic sympathomimetic activity (ISA) or partial agonist activity. Each subject received each drug and a placebo for 1 wk each according to a randomized double-blind crossover design. After placebo, exercise caused 10-20% increases in total plasma cholesterol and the high-density lipoprotein (HDL)-cholesterol fraction. The total-to-HDL cholesterol ratio fell, particularly during the 30-min recovery phase. Pindolol treatment increased resting values of HDL cholesterol (from 43 +/- 4 to 48 +/- 4 mg/dl) and potentiated the response to exercise (to 59 +/- 5 vs. 51 +/- 4 mg/dl after placebo). The total-to-HDL cholesterol ratio was significantly lower after pindolol treatment than after placebo. In contrast, neither atenolol nor timolol affected exercise-induced changes in plasma lipoprotein levels. The effects of pindolol on other study parameters (exercise endurance and exercise-induced increases in systolic blood pressure, heart rate, and potassium) were similar to the effects of the nonselective agent, timolol. We conclude that the effects of pindolol on the plasma lipid profile are due to its ISA and that the process activated (possibly plasma lecithin-cholesterol acyltransferase activity) is under minimal sympathetic control and, therefore, sensitive to the expression of ISA both at rest and in response to exercise.     

Substitution in vitro of lecithin-cholesterol acyltransferase. Analysis of changes in plasma lipoproteins

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified 15 000-fold from human plasma. The active material was homogeneous in different gel electrophoretic systems but separated into three major bands with apparent pI values of 4.28, 4.33 and 4.37 in isoelectrofocusing. The apparent Mr of the enzyme is 67 000 +/- 2000. An antiserum prepared against the purified enzyme specifically inhibited the activity of lecithin-cholesterol acyltransferase in whole serum. Serum from a patient with familial deficiency of lecithin-cholesterol acyltransferase was substituted in vitro with the highly purified enzyme. The serum from this patient did not contain immunochemically detectable enzyme protein. Substitution of enzyme resulted in the following major changes. 1. Cholesteryl ester content in serum increased by 36-89 mg/100 ml depending on the experimental conditions. The enzyme-mediated formation of cholesteryl ester led to an increase of cholesteryl ester content in high-density and very-low-density lipoproteins and in low-density lipoproteins containing apoprotein-B. No increase occurred in fractions containing very large flattened structures and the abnormal lipoprotein-X and in lipoprotein-E. Incubation of isolated fractions with lecithin-cholesterol acyltransferase led to significant cholesterol esterification only in high-density lipoproteins. 2. The characteristic disc-shaped rouleaux-forming high-density lipoproteins of enzyme-deficient serum disappeared. Instead a single homogeneous population of high-density lipoproteins formed. The particles generated were spherical and had the electrophoretic properties, density (1.080 g/ml), diameter (12.5 nm) and apoprotein composition of normal high-density lipoproteins-2. 3. The concentration of spherical particles containing apolipoprotein E (density 1.040-1.080 g/ml) and the lamellar lipoprotein-X-like structures in the low-density lipoprotein fraction were not affected by the enzyme substitution. 4. A single homogeneous population of spherical lipoprotein-B particles of 26.5-nm diameter occurred at density 1.029 g/ml. The data suggest that the discoidal high-density lipoproteins are the major site of cholesteryl ester formation that apolipoprotein-E is not involved in an undirectional transport of newly formed cholesteryl ester from high-density lipoproteins to other lipoproteins and that lipoprotein-X and lipoprotein-E are not preferential substrates for the acyltransferase.     

Renal parathyroid hormone-dependent adenylate cyclase in vitamin D-deficient rats. Inhibition by hydroxylated vitamin D3 metabolites

The adenylate cyclase activation by bovine synthetic parathyroid hormone (bPTH) (1-34) was studied in vitro in kidney plasma membranes from D-deficient (D-Mb) or normal (D+Mb) rats. In D-Mb, the apparent affinity of parathyroid hormone (PTH) for membranes (170 +/- 30 nM) was significantly higher than that measured in D+Mb (55 +/- 5 nM). The maximum velocity of the PTH-stimulated adenylate cyclase was significantly higher in D+Mb than in D-Mb (163.0 +/- 13.7 and 93.4 +/- 6.7 pmol of cAMP/mg of protein/min, respectively). The action of vitamin D metabolites on the adenylate cyclase stimulation by PTH was then studied in vitro in D-Mb and D+Mb. In D-Mb, 25-hydroxyvitamin D3, 24,25-, and 1, 25-dihydroxyvitamin D3 significantly inhibited cAMP production in the presence of 0.87 microM of bPTH. Vitamin D3 had no effect. Maximal inhibition (86%) was observed for 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 decreased the maximum velocity of PTH-stimulated adenylate cyclase but did not modify the bPTH apparent affinity for D-Mb. The vitamin D3 metabolites tested did not modify the cyclase stimulation by isoproterenol, sodium fluoride, or 5'-guanylylimidodiphosphate. The presence of 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 did not increase the (Na-K)-ATPase or the phosphodiesterase activities. In the presence of 1,25-dihydroxyvitamin D3 and bPTH, the apparent affinity of ATP for the catalytic moiety was not modified. The maximum velocity was decreased. These results suggest an in vitro interaction between hydroxylated vitamin D metabolites and kidney membranes PTH receptor.     

Changes in the surface charge of plasma lipoproteins during their auto-oxidation

Low density lipoproteins (LDL) and high density lipoproteins (HDL) surface potential (charge) changes were studied upon autooxidation, using positively charged spin probes. Lipid peroxidation product accumulation in LDL and HDL suspensions was found to be accompanied by a significant reduction in their surface area associated with a decreased negative surface charge, and probably, deposition of lipid peroxidation polar products and/or surface charge redistribution as a result of lipoprotein autooxidative modification.     

Alterations of the gamma-carboxyglutamic acid and osteocalcin concentrations in vitamin D-deficient chick bone

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.     

Structural-functional changes in the synaptic membranes of the cerebral cortex of rats during electric stimulation in vitro]

Electric stimulation (EC) of a suspension of native synaptic membranes of rat brain cortex in the Krebs-Ringer-glucose medium revealed Ca-dependent inhibition of Na+, K+-ATPase and inhibition of transport Ca-activated, Mg-dependent ATPase. The effects observed are not induced by a change in the SH-groups of the membrane proteins and are removed by an addition of total lipids of the brain (membrane protein: lipid = 5:1) or 0.35 mM novocaine. Cyclic 3',5'-AMP in concentrations of 0.1--1.0 mM causes an inhibition (up to 50%) of Na+, K+-ATPase of native synaptic membranes. The Na+, K+-ATPase activity of purified membrane preparations is not changed either by the cyclic nucleotide, or by EC. It is assumed that depolarization of excitable membranes results in structural changes, mediated by the activation of protein kinase, and manifesting themselves as labilization of protein-lipid ratios.