Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma.

Multiple myeloma is a B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow and the development of osteolytic bone disease. The present study demonstrates that myeloma cells express the critical osteoclastogenic factor RANKL (the ligand for receptor activator of NF-kappa B). Injection of 5T2MM myeloma cells into C57BL/KaLwRij mice resulted in the development of bone disease characterized by a significant decrease in cancellous bone volume in the tibial and femoral metaphyses, an increase in osteoclast formation, and radiologic evidence of osteolytic bone lesions. Dual-energy x-ray absorptiometry demonstrated a decrease in bone mineral density (BMD) at each of these sites. Treatment of mice with established myeloma with recombinant osteoprotegerin (OPG) protein, the soluble decoy receptor for RANKL, prevented the development of lytic bone lesions. OPG treatment was associated with preservation of cancellous bone volume and inhibition of osteoclast formation. OPG also promoted an increase in femoral, tibial, and vertebral BMD. These data suggest that the RANKL/RANK/OPG system may play a critical role in the development of osteolytic bone disease in multiple myeloma and that targeting this system may have therapeutic potential.

Cereal grains, alpha tocotrienol and cholesterol metabolism in the rat.

The influence of alpha (α)-tocotrienol, the main vitamer of vitamin E in barley and oats, on cholesterol synthesis has been studied in laboratory rats. Both oats and barley lowered plasma cholesterol relative lo wheat, which had no such effect, and the change has been attributed to an inhibitory influence of a -tocotrienol on cholesterol synthesis rate. Vitamin E was stripped from oats and barley by a petroleum ether extraction procedure and the grains compared with their unstripped equivalents. In the oats feeding experiment this resulted in a higher plasma cholesterol and lower liver cholesterol synthesis rate. The barley experiment produced no significant response. Pure α-tocotrienol was gavaged into rats fed a semipurified diet without vitamin E, at the rate of 380 µg/rat/day for 28 days. There was no significant influence on plasma cholesterol level or on liver cholesterol synthesis rate. From these studies it is concluded that a -tocotrienol does not influence cholesterol synthesis rate significantly. Therefore, it is unlikely lo be a factor in oats and barley responsible for the plasma cholesterol lowering observed.

Effects of solvent extraction on the hypocholesterolaemic action of oat bran in the rat.

In adult male rats fed on a cholesterol-free synthetic diet, plasma cholesterol concentrations were lowest with oat bran, intermediate with cellulose and highest with wheat bran. Plasma triacylglycerols (TAG) were similar with wheat bran and cellulose but higher with oat bran. The concentrations and pools of caecal volatile fatty acids (VFA) were lowest with cellulose and equally higher with oat bran and wheat bran. Plasma VFA concentrations in the hepatic portal vein reflected those in caecal digesta and were unrelated to plasma cholesterol. Feeding oat bran after extraction with n-pentane gave plasma cholesterol concentrations similar to that found with wheat bran. Reconstitution of oat bran with extracted lipids did not restore the cholesterol-lowering effect. Addition of the extracted material to a wheat-bran diet had no effect on plasma cholesterol. Plasma TAG were higher with the oat bran and reconstituted-oat-bran diets than with wheat-bran or cellulose diets. However, extracted oat bran + safflower oil gave similar TAG concentrations to that with wheat bran. These extractions and additions did not change caecal bile acid or neutral sterol concentrations. Effects of these diets on plasma cholesterol were unrelated to their tocotrienol or tocopherol content. Addition of n-pentane to oat bran followed by evaporation of solvent gave plasma cholesterol concentrations that were significantly higher than untreated oat bran but lower than similarly treated wheat bran. It is concluded that oat bran affects cholesterol metabolism through a pentane-soluble component as well as non-starch polysaccharides. It appears that the activity of this lipid is not transferable by simple addition of the solvent extract to the whole diet.

The influence of linoleate and vitamin E from sunflower seed oil on platelet function and prostaglandin production in the common marmoset monkey.

Vitamin E and linoleate, both of which are found in high concentrations in sunflower seed oil, were examined independently for their influence on general and blood-vascular parameters in vitamin E-deficient common marmosets. A vitamin E-deficient diet (-E, 4 micrograms/g) was supplemented with either 40 micrograms/g vitamin E (+E), vitamin E stripped sunflower oil (+10% SSO-E), or SSO (+10% SSO w/w) in a 2 x 2 factorial designed experiment, and the diets fed for 9 months to 4 even groups of common marmosets. Vitamin E deficiency was associated in marmosets with a loss of skeletal muscle mass and of body weight, enhanced peroxidative haemolysis of erythrocytes, increased white blood cell counts, and in the SSO-E group a relative neutrophilia. Platelet reactivity was increased with vitamin E deficiency, and to a greater degree with the SSO-E group. Aortic prostacyclin production was significantly increased by the addition of vitamin E, linoleate and both as SSO to the deficient diet, the effects being additive. Fatty acid changes associated with the different treatments reflected the influence of high linoleate and vitamin E treatments. The platelet and aortic arachidonate value in the SSO-E group showed the lowest and most variable value, and this was associated with greatest platelet aggregability. An adequate vitamin E intake is essential for stabilising high PUFA diets and biomembranes and enhancing the protective role of prostacyclin in blood vessels against thrombogenesis.

Changes in fatty acid composition of the cardiac phospholipids of the cotton-eared marmoset (Callithrix jacchus) after feeding different lipid supplements.

After feeding marmosets different lipid supplements for 6 months, the distribution of phospholipid classes and the fatty acid composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) were determined in their cardiac membranes. Supplementing the diet with linoleic-acid-rich sunflower seed oil raised the level of 18:2,n-6 in both PC and PE, but did not change the level significantly in DPG. When 18:2,n-6 was increased, the level of arachidonic acid (20:4,n-6) was significantly decreased in PC and PE. No arachidonic acid was present in DPG. Supplementing the diet with mutton fat did not markedly increase the level of saturated fats, nor did it markedly reduce the level of arachidonic acid in any phospholipid component. No dietary treatment altered the distribution of the major phospholipid classes.

Altered levels of n-6/n-3 fatty acids in rat heart and storage fat following variable dietary intake of linoleic acid.

Fat-supplemented dies enriched with linoleic acid by the addition of 12% w/w sunflower seed oil or proportionally reduced in linoleic acid by addition of 12% mutton fat were fed to rats for 18 months before the fatty acid composition of perirenal storage fat and myocardial membranes (phospholipids) was determined. Although the fatty acid composition of perirenal fat generally reflected that of the diet, there was an inverse relationship between the consumption of n-6 and the deposition of n-9 fatty acids. In addition, enhanced deposition of oleic acid (18:1, n-9) appears to be related to the dietary intake of stearic acid (18:0). In contrast, in myocardial membranes the n-3 polyunsaturated fatty acids are found to be increased when the intake of n-6 polyunsaturated fatty acids is reduced. This is particularly evident for docosahexaenoic acid (22:6, n-3) which is significantly increased in phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol fractions of myocardial membranes, when the mutton fat diet was fed. After feeding the sunflower seed oil diet, the increased consumption of linoleic acid produced only small changes in the 18:2, n-6 content of cardiac phosphatidylcholine and phosphatidylethanolamine. These major classes of membrane phospholipids also showed only small increases in 20:4, n-6. In diphosphatidylglycerol, increased 18:2, n-6 also followed increased dietary intake, but this was not accompanied by increased 20:4, n-6. These changes in myocardial phospholipid fatty acid composition are similar to those observed after short-term feeding reported previously and confirm that changes in dietary n-6/n-3 fatty acid intake affect the fatty acid composition of both myocardial membranes and storage fat. These changes persist for the duration of the feeding period.

Species variation in the ouabain sensitivity of cardiac Na+/K+-ATPase. A possible role for membrane lipids.

The role of membrane lipid composition on the modulation of ouabain sensitivity of cardiac Na+/K+-ATPase has been studied in vitro using several animal species. The animals can be grouped as ouabain-sensitive and ouabain-insensitive species. Ouabain-sensitive species (I50; 0.5-2.2 microM) include sheep, marmoset, pig and the guinea pig, whilst rat and mouse form the ouabain-insensitive group (I50; 100-105 microM). Although no species variation in the distribution of major phospholipid classes was observed, significant differences were apparent in the proportions of certain saturated and unsaturated phospholipid fatty acids. Thus, there was a marked increase in the relative proportion of docosahexaenoic (22:6, omega-3) acid in the Na+/K+-ATPase preparations from the rat and mouse compared to ouabain-sensitive species. Despite these differences, all animals had similar proportions of total saturated (sigma SAT) and total unsaturated (sigma Unsat) fatty acids. On the other hand, a good correlation between the unsaturation index of membrane lipids and I50 value for ouabain was observed. It is proposed that acyl chain characteristics (unsaturation and/or chain length) rather than the head group of the phospholipid molecule play a major role in the modulation of Na+/K+-ATPase to inhibition by ouabain.

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid.

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na+ + K+)ATPase and sarcoplasmic reticulum Ca2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n-6 fatty acids, including linoleic (18:2(n-6] and arachidonic (20:4(n-6] while the sheep kidney fat dietary rats were higher in n-3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n-6/n-3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.

The composition of cardiac phospholipids in rats fed different lipid supplements.

Changes in dietary lipid intake are known to alter the fatty acid composition of cardiac muscle of various animals. Because changes in cardiac muscle membrane structure and function may be involved in the pathogenesis of arrhythmia and ischemia, we have examined the effects of dietary lipid supplements on the phospholipid distribution and fatty acid composition of rat atria and ventricle following 20 weeks feeding of diets supplemented with either 12% sunflower-seed oil or sheep fat. Neither lipid supplement produced significant changes in the proportions of cholesterol, total phospholipids or phosphatidylcholine, phosphatidylethanolamine or diphosphatidylglycerol,--the phospholipid classes that together account for more than 90% of the total phospholipids of rat cardiac muscle. Significant changes were found in the profiles of the unsaturated fatty acids of all 3 phospholipid components of both atria and ventricle. Although similar, the changes between these tissues were not identical. However, in general, feeding a linoleic acid-rich sunflower seed oil supplement resulted in an increase in the omega-6 family of fatty acids, whereas feeding the relatively linoleic acid-poor sheep fat supplement decreased the level of omega-6 fatty acids but increased the levels of the omega-3 family, resulting in major shifts in the proportions of these families of acids. In particular, the ratio of arachidonic acid: docosahexaenoic acid (20:4,omega-6/22:6,omega-3), which is higher in all phospholipids of atria than ventricle, is increased by feeding linoleic acid, primarily by increasing the level of arachidonic acid in the muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the fatty acid composition of atrial and ventricular phospholipids of rat heart following standard and lipid-supplemented diets.

1. The major saturated fatty acids of the phospholipids of rat heart atria and ventricles are similar and are not greatly altered by supplementing the diet with widely different types of lipid. 2. There are important differences in the relative proportions of the major unsaturated fatty acids of the phospholipids of these anatomically and functionally distinct regions of the heart. 3. The proportions of linoleic (C18:2, eta-6) and docosahexaenoic (C22:6, eta-3) acid are significantly higher in the ventricles than in the atria; the proportions of oleic (C18:1, eta-9) arachidonic (C20:4, eta-6) and docosatetraenoic acids (22:4, eta-6) are higher in atria. 4. The differences in unsaturated fatty acid profiles persist even after twelve months of feeding lipid supplements of sunflower seed oil (SSO) or sheep kidney (perirenal) fat (SKF). 5. However, the ratios of arachidonic to docosahexaenoic acid in both tissues are changed by decreasing the intake of linoleic acid, which apparently favours the conversion of dietary linolenic (C18:3, eta-3) to docosahexaenoic acid. The level of docosahexaenoic acid is greater in the ventricles than in the atria, and greatest when the animals were fed SKF diet. 6. The physiological and pharmacological differences in ventricles and atria may arise from differences as fundamental as the phospholipid fatty acid composition of cardiac membranes.

Effects of variations in nucleoside pool sizes on comparisons of the incorporation of [3H]thymidine into isolated rat liver cells.

Isolated rat liver cells have been used to measure DNA repair synthesis induced by ultraviolet light and aflatoxin. Age, sex, and diet of the rats were found to influence DNA repair in rat liver cells, as measured by [3H]thymidine uptake by the cells. These effects were found to be due to variations in the thymidine pool size and should serve to indicate that high-specific-activity thymidine (i.e., low nucleoside concentrations) is subject to artifacts generated by pool size variations.

Induction of DNA repair by some selenium compounds.

Selenium compounds were found to induce DNA repair synthesis as a measure of DNA damage in both the isolated rat liver cell system and by Ames' Salmonella assay. In liver cells, DNA repair measured by uptake of [3H]thymidine was found to be greater with sodium selenite and selenate than with selenomethionine. In the bacterial culture system, selenomethionine inhibited the repair-deficient variant more than the selenite and selenate. These in vitro test systems have been used to indicate that selenium has a DNA-damaging potential.

Amino acid nutrition of the fetal lamb.

With three exceptions amino acids were retained by the fetal lamb from the maternal circulation in proportions appropriate for the synthesis of whole blody protein. Only glutamate, aspartate and serine, amino acids involved in the synthesis of nucleic acids, seemed to be produced by the fetus itself.

Methylmalonic acid and coenzyme A concentrations in the livers of pair-fed vitamin B 12-deficient and vitamin B 12-treated sheep.

The concentrations of CoA in the livers of severely vitamin B(12)-deficient ewes were about 2.6 times those in pair-fed animals treated with vitamin B(12). When the feeding rates of the pair-fed animals were closely similar, the concentrations of methylmalonic acid in deficient livers were about twice those in vitamin B(12)-sufficient livers. The molar concentrations of CoA present were more than three times those of methylmalonic acid in both deficient and treated animals, and it is concluded that the elevated concentrations of CoA in the deficient livers were not primarily due to accumulation of methylmalonyl-CoA.

Metabolism of propionate by sheep liver. Pathway of propionate metabolism in aged homogenate and mitochondria.

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-(14)C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of (14)C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of (14)C in dicarboxylic acids, citrate, alpha-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-(14)C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.

Metabolism of propionate by sheep-liver mitochondria. Effects of alpha-oxoglutarate, adenosine triphosphate, sodium chloride and potassium chloride.

1. A study has been made of the effects of ATP and alpha-oxoglutarate on the rate of metabolism of propionate by whole mitochondria from sheep liver, and by mitochondria disrupted with ultrasonic energy or by freezing and thawing. Whole mitochondria metabolized propionate aerobically; the rate was increased and stabilized by 0.5mm-ATP, and increased at least a further 50% by 1.67mm-alpha-oxoglutarate. 2. Anaerobically, externally added ATP at high concentrations permitted slow consumption of propionate. 3. In the presence of 1.3mm-ATP, but in the absence of alpha-oxoglutarate, there was no significant lag phase in the removal of propionate by whole mitochondria, and the rate declined at concentrations below 2mm. In the additional presence of 1.67mm-alpha-oxoglutarate or -glutamate, propionate was removed at linear rates until the residual propionate concentration was about 0.1mm. 4. Maximum rates of metabolism of propionate by whole mitochondria with 1.3mm-ATP occurred with alkali-metal chloride concentrations of 65-95mm and with K(+)/Na(+) ratios 5-10, both in the presence and absence of alpha-oxoglutarate. 5. With disrupted mitochondria stimulatory effects of alpha-oxoglutarate were obtained only aerobically, only with propionate and not propionyl-CoA as substrate, and only when sufficient mitochondrial structure remained to permit unsupplemented metabolism of propionate to occur. 6. In the presence of ATP and CoA, disrupted mitochondria fixed [2-(14)C]propionate at a rate adequate to explain the rate with whole mitochondria stimulated with ATP and alpha-oxoglutarate. 7. With both whole and partially disrupted mitochondria in the absence of ATP, the rate of metabolism of propionate was inhibited by about 80% by 3.3mm-AMP. The inhibition was partly overcome by alpha-oxoglutarate plus CoA. 8. It is concluded that the ultimate effect of alpha-oxoglutarate was to increase the rate of supply of ATP within the mitochondria. Reasons are given why it is premature to conclude that the extra ATP arose entirely from the oxidation of alpha-oxoglutarate itself.

Metabolism of propionate by sheep-liver mitochondria. Evidence for rate control by a specific succinate oxidase.

1. Metabolism of propionate by sheep-liver mitochondria was stimulated catalytically by alpha-oxoglutarate, pyruvate, citrate and isocitrate. Succinate was stimulatory at higher concentrations, but fumarate and malate were inert. These effects were all independent of the presence of ATP, succinate being less effective when ATP was present. 2. Compared with the metabolism of added succinate, propionate metabolism was resistant to malonate inhibition, but only in the presence of added ATP. In the absence of ATP propionate metabolism was more sensitive to malonate inhibition than was the metabolism of succinate. 3. In the absence of malonate, and at malonate concentrations in the range 5-100mm, alpha-oxoglutarate increased the rate of fixation of [2-(14)C]propionate by about 50% without altering the nature of the fixation products. 4. Metabolism of [1-(14)C]-propionate in the presence of 50mm-malonate was accompanied by accumulation of about half the propionate consumed as succinate. When alpha-oxoglutarate was present in addition part of the alpha-oxoglutarate was metabolized and the rate of propionate consumption was increased. The total succinate that accumulated corresponded to the alpha-oxoglutarate consumed plus about half the propionate metabolized. 5. When [1-(14)C]propionate was metabolized in the absence of malonate about 70% of the generated succinate was oxidized to fumarate or beyond. The addition of malonate decreased the rate of propionate metabolism, and decreased to about half the fraction of generated succinate oxidized. 6. When propionate and 10mm-succinate were metabolized together, the total oxidation of succinate was greater than that with 10mm-succinate alone. The increment in succinate oxidation corresponded to about half the propionate metabolized in the presence or absence of malonate or ATP. 7. It is suggested that the metabolism of propionate is specifically limited by the rate of oxidation of the generated succinate, and that the succinate oxidase concerned is distinct from that responsible for the oxidation of added succinate. 8. The results are discussed in terms of the mode of action of certain stimulants and inhibitors of propionate metabolism. It is suggested that many of these act by stimulation or inhibition of the specific succinate oxidase that limits propionate metabolism.