Kenneth G.D. Allen

Dietary Conjugated Linoleic Acids Alter Serum IGF-I and IGF Binding Protein Concentrations and Reduce Bone Formation in Rats Fed (n-6) or (n-3) Fatty Acids†

A study was designed to examine the effects of dietary conjugated linoleic acid (CLA) on serum concentrations of insulin‐like growth factor‐I (IGF‐I) and IGF binding proteins (IGFBP) and the relationship of these factors to bone metabolism. Weanling male rats were fed AIN‐93G diet containing 70 g/kg of added fat for 42 days. Treatments included 0 g/kg or 10 g/kg of CLA and soybean oil (SBO) or menhaden oil + safflower oil (MSO) following a 2 × 2 factorial design. Serum IGFBP was influenced by dietary polyunsaturated fatty acid (PUFA) type ((n‐6) and (n‐3)) and CLA (p = 0.01 for 38–43 kDa bands corresponding to IGFBP‐3). CLA increased IGFBP level in rats fed SBO (p = 0.05) but reduced it in those fed MSO (p = 0.01). Rats fed MSO had the highest serum IGFBP‐3 level. Both (n‐3) fatty acids and CLA lowered ex vivo prostaglandin E2 production in bone organ culture. In tibia, rats given CLA had reduced mineral apposition rate (3.69 vs. 2.79 μm/day) and bone formation rate (BFR) (0.96 vs. 0.65 μm3/μm2/day); however, the BFR tended to be higher with MSO. Dietary lipid treatments did not affect serum intact osteocalcin or bone mineral content. These results showed that dietary PUFA type and CLA modulate local factors that regulate bone metabolism.

The Role of n-3 Fatty Acids in Gestation and Parturition

Preterm birth is the most common cause of low infant birth weight and infant morbidity and mortality. Evidence from human and animal studies indicates that essential fatty acids of both the n-3 and n-6 series, and their eicosanoid metabolites, play important and modifiable roles in gestational duration and parturition, and n-3 fatty acid intake during pregnancy may be inadequate. Prostaglandins (PG) of the 2-series are involved in parturition and connective tissue remodeling associated with cervical maturation and rupture of membranes. In the absence of infections, preterm birth is characterized by lower reproductive tissue PG production and decreased inducible cyclooxygenase expression. Women who deliver prematurely have increased pools of n-6 fatty acid and decreased n-3 fatty acids, despite the lower PG production. Several human pregnancy supplementation trials with n-3 fatty acids have shown a significant reduction in the incidence of premature deliver and increased birth weight associated with increased gestational duration. Supplementation with long chain n-3 fatty acids such as docosahexaenoic acid may be useful in prolonging the duration of gestation in some high-risk pregnancies. Evidence presented in this review is discussed in terms of the roles of dietary n-3 and n-6 fatty acids in gestation and parturition, mechanisms by which they may influence gestational duration and the human trials suggesting that increased dietary long-chain n-3 fatty acids decrease the incidence of premature delivery.

Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia.

Dietary copper deficiency causes hypercholesterolemia and increased hepatic 3‐hydroxy‐3‐methylglutaryl coenzyme A (MHG‐CoA) reductase activity and increased hepatic glutathione (GSH) in rats. We hypothesized that inhibition of GSH production by l‐buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor, would abolish the cholesterolemia and increased HMG‐CoA reductase activity of copper deficiency. In two experiments, two groups of 20 weanling male rats were fed diets providing 0.4 and 5.8 μg Cu/g, copper‐deficient (Cu‐D) and copper‐adequate (Cu‐A), respectively. At 35 days plasma cholesterol was significantly elevated by 30 to 43% in Cu‐D and 10 animals in each of the Cu‐D and Cu‐A groups were randomly assigned to receive 10 mm BSO solution in place of drinking water and continued on the same diets for another 2 wk. At necropsy Cu‐D animals had a significant 52 to 58% increase in plasma cholesterol. BSO administration abolished the cholesterolemia in Cu‐D rats, but had no influence on plasma cholesterol of Cu‐A rats. Hepatic GSH was increased 39 to 82% in Cu‐D rats and BSO abolished this increase. BSO was without effect on cardiac hypertrophy, plasma and liver copper, and hematocrit indices of copper status. Liver microsome HMG‐CoA reductase activity was significantly increased 85 to 288% in Cu‐D rats and BSO administration abolished this increase in activity in Cu‐D rats. The results suggest that copper deficiency cholesterolemia and elevated HMG‐CoA reductase activity are a consequence of elevated hepatic GSH, and provide evidence for GSH regulation of cholesterol metabolism in intact animals.—Kim, S.; Chao, P. Y.; Allen, K. G. D. Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia. FASEB J. 6: 2467‐2471; 1992.

The Omega-3 Story:: Nutritional Prevention of Preterm Birth and Other Adverse Pregnancy Outcomes

Healthy intrauterine growth and development, appropriately timed parturition, and safe birth continue to be critical determinants of each child’s chances of achieving their full biological potential. Essential fatty acids (EFAs) and their metabolites are involved in the synthesis of eicosanoids that play essential and multifaceted roles in human reproduction, especially during pregnancy, parturition, and lactation. Fatty acid-derived prostaglandins (PGs), hydroxyeicosatetraenoic molecules (HETE), leukotrienes (LTs), thromboxanes (TXs), and prostacyclins (PIs) play well-documented “final common pathway” molecular roles in both normal term labor as well as in preterm parturition (1). Abnormalities of thromboxane and prostacyclin physiology also occur with preeclampsia, eclampsia, and pregnancy-induced hypertension (PIH), which often require preterm delivery (2). Docosahexaenoic acid (22: 6v-3, DHA) and other EFA derivatives are important components of the rapidly growing brain and retinal tissues of the perinate (3). DHA, a long-chain omega-3 (v-3) fatty acid (LCPUFA) is expressed in breast milk, and breast-fed and DHA-nutritionally supplemented children demonstrate improved performance on various tests of brain and vision functioning (4). Ensuring maternal and perinatal nutritional sufficiency during pregnancy and lactation is investigated increasingly as an effective and inexpensive strategy for optimizing pregnancy and lactational outcome (5). Informed families and providers are invested both in preventing the occurrence and consequences of prematurity and inadequate uterine growth (6). Families and providers are similarly interested in optimizing healthy growth and development of the fetus’ developing organs, especially brain, retina, and the cardiovascular system (7). Preterm birth continues to cause neonatal morbidity and life time disabilities (8). Preterm birth is associated with potentially immense direct and indirect costs, as well as liabilities for care providers. Preterm birth is best understood as a syndrome caused by numerous diseases such as intrauterine infection and inflammation, uterine over-distention or intrauterine bleeding. Such causes of prematurity are best considered as “complex diseases.” Complex diseases involve interactions between multiple bodily systems or parameters including nutrition, immunity, coagulation, and the endocrine system. For example, the fetus depends on maternal nutrients, such as EFAs, for multiple bodily functions including eicosanoid metabolism as well as brain growth and retinal development. Thus, if the mother is deficient in certain fatty acids, this deficiency tends to be compounded in the fetus (9). Such complex disease processes are best dealt with using primary prevention techniques—such as ensuring nutritional sufficiency, particularlyv-3 LCPUFA sufficiency, during pregnancy and lactation.

Omega-3 fatty acids enhance ligament fibroblast collagen formation in association with changes in interleukin-6 production.

Altering dietary ratios of n-3 and n-6 polyunsaturated fatty acids (PUFA) represents an effective nonpharmaceutical means to improve systemic inflammatory conditions. An effect of PUFA on cartilage and bone formation has been demonstrated, and the purpose of this study was to determine the potential of PUFA modulation to improve ligament healing. The effects of n-3 and n-6 PUFA on the in vitro healing response of medial collateral ligament (MCL) fibroblasts were investigated by studying the cellular coverage of an in vitro wound and the production of collagen, PGE2, IL-1, IL-6, and TNF. Cells were exposed to a bovine serum albumin (BSA) control or either eicosapentaenoic acid (EPA, 20:5n-3) or arachidonic acid (AA, 20:4n-6) in the form of soaps loaded onto BSA for 4 days and wounded on Day 5. AA and EPA improved the healing of an in vitro wound over 72 hr. EPA increased collagen synthesis and the overall percentage of collagen produced, but AA reduced collagen production and total protein. PGE2 production was increased in the AA-treated group and decreased in the EPA-treated group, but was not affected by wounding. IL-1 was not produced at the time point evaluated, but TNF and IL-6 were both produced, and their levels varied relative to the PUFA or wounding treatment. There was a significant linear correlation (r2 = 0.57, P = 0.0045) between IL-6 level and collagen production. These results demonstrate that n-3 PUFA (represented by EPA in this study) positively affect the healing characteristics of MCL cells and therefore may represent a possible noninvasive treatment to improve ligament healing. Additionally, these results show that MCL fibroblasts produce PGE2, IL-6, and TNF and that IL-6 production is related to MCL collagen synthesis.

Stearidonic and Eicosapentaenoic Acids Inhibit Interleukin-6 Expression in ob/ob Mouse Adipose Stem Cells via Toll-Like Receptor-2–Mediated Pathways

Increased adipose tissue positively correlates with circulating inflammatory cytokines such as IL-6. We previously reported that adipose stem cells from genetically obese ob/ob mice produce significantly higher levels of IL-6 compared with other cell types such as adipocytes and macrophages within adipose tissue. We also demonstrated that (n-3) PUFA have antiinflammatory effects on adipocyte IL-6 secretion. Based on these findings, we hypothesized that EPA [20:5 (n-3)] and stearidonic acid [SDA, 18:4 (n-3)] would decrease LPS (200 μg/L)-induced IL-6 secretion and IL-6 mRNA content in the adipose stem cells. SDA (100 μmol/L) and EPA (100 μmol/L) significantly reduced LPS-induced IL-6 secretion and decreased IL-6 mRNA expression. To determine the underlying intracellular mechanisms, we tested whether LPS-induced Toll-like-receptor (TLR) 4 and TLR2 expression were modulated by these fatty acids using Western-blot analysis. EPA and SDA suppressed LPS-induced TLR2 but not TLR4 protein expression in the adipose stem cells. Furthermore, SDA and EPA significantly lowered the activation and translocation of NF-κB, a TLR2 downstream signaling target, while protein expression of extracellular signal-regulated kinases-1/2 were unaffected. Collectively, our results suggest that EPA and SDA inhibit LPS-induced IL-6 secretion and IL-6 mRNA expression in the adipose stem cells by decreasing TRL2-mediated signaling pathways.

Marginal copper deficiency in rats aortal morphology of elastin and cholesterol values in first-generation adult males

A marginal, 2 parts per million (ppm) copper diet (experimental) was fed to female rats for 4 months prior to breeding, through gestation/lactation, and to the weaned offspring to determine the consequences in adult, male offspring on cholesterol values and aortal morphology. Liver copper concentrations of the dams and pups at day 21 of lactation and of the 117-day-old offspring who consumed the experimental diet were lower (P less than 0.0001) than corresponding rats fed a 10 ppm copper diet (control). However, statistically significant differences due to dietary treatments were not evident in pre- or post-weaning gain in body weight, litter size, cannibalism of pups, or total cholesterol concentrations of the serum and aorta. Ultrastructural examination of experimental offspring aortas revealed focally abnormal features of endothelial cells, the subendothelial space, collagen fibers, smooth muscle cells, and particularly elastin. The ultrastructural irregularities of elastin included discontinuous regions of the internal elastic lamina comprised of stained clumps of elastin of irregular size and shape. The results of this study suggest that a marginal copper nutriture begun in utero will elicit morphologic abnormalities of the aorta in rats that are otherwise without overt signs of copper deficiency.

Oxidized lipid depresses canine growth, immune function, and bone formation.

Dietary oxidized lipids can increase oxidative stress and potentially contribute to a variety of disease syndromes. This research describes the first use of a canine model to assess the effects of dietary oxidized lipids on growth, antioxidant status, and some immune functions. Three groups of eight, two-month old coon-hound puppies were pair fed diets for 16 weeks. The control diet contained <50 ppm aldehydes, and two additional diets contained thermally oxidized lipids targeted to contain 100 ppm aldehydes (medium-oxidation) and 500 ppm aldehydes (high-oxidation). Dogs fed the high-oxidation diet weighed less than those from the medium-oxidation (P < 0.05) and control groups (P < 0.001) at the end of the study. Oxidized lipids reduced serum vitamin E levels, total body fat content, and bone appositional rate. At different time points of the study, peripheral blood neutrophils and monocytes from dogs fed the HO diet had reduced oxidative burst capacity and produced less superoxide and hydrogen peroxide when stimulated with phorbol esters compared to the control group. Lymphocyte blastogenesis in response to concanavalin A was suppressed by dietary oxidized lipid. This study indicates that dietary oxidized lipids negatively affect the growth, antioxidant status, and some immune functions of dogs. Importantly, some effects are evident at 100 ppm aldehydes in the diet, which is a moderate level of oxidation. The rapid growth and weight gain of the dog during the first 6 months of life may also provide a better model for assessing the risks of dietary oxidized lipid in children and adolescents than previously used rodent models.

Enzymatic modification to improve the water-absorbing and gelling properties of psyllium

Psyllium husks were treated with a commercial food-grade polysaccharidase mixture, under solid-state reaction conditions, to improve water-absorbing and gelling properties. The modified psyllium preparations were analyzed and compared to the original psyllium and the control, treated with no enzyme under the same reaction conditions, for their water-absorbing ability, gelling properties, fibre contents, and surface structures. The water-absorbing ability was determined by a gravimetric method, while the gelling property of the modified psyllium was measured using a texture analyzer. The results showed that the solid-state enzymatic modification was able to significantly reduce both water-absorbing and gelling abilities of psyllium. Compared to the control, reductions of 49% in water-absorbing ability, 71% in gel hardness, and 35% in gel adhesiveness were observed for the novel psyllium preparation that was treated with the enzyme mixture at a level of 36 units/g psyllium. The surface structure of the modified psyllium was examined using scanning electron microscopy (SEM). The SEM results showed that the enzymatic modification decreased the total surface area. This may contribute to the reduced water-absorbing ability of the modified psyllium. This study demonstrated the potential of preparing novel psyllium preparations, using a solid-state enzymatic method, for commercial food applications.

Copper deficiency depresses rat aortae superoxide dismutase activity and prostacyclin synthesis

Prostaglandin synthesis shows dependence on lipid hydroperoxides and resultant oxygen derived radical formation. In view of the importance of dietary copper in cytosolic copper dependent superoxide dismutase (Cu/Zn SOD) activity and the role of SOD in oxygen radical formation, the influence of dietary copper on prostacylin (PGI2) synthesis and SOD activity in rat aorta was examined. Copper deficient (0.5 micrograms Cu/g diet) rats showed a significant 47% reduction in PGI2 synthesis rates by aortic ring incubations in comparison to copper adequate (6.0 micrograms Cu/g diet) animals. Aortic SOD activity was reduced by 46% in copper deficiency in comparison to copper adequate animals. Marginal dietary copper (1.6 micrograms Cu/g diet) significantly reduced aortic SOD activity by 32% but was without effect on aortic ring incubation PGI2 synthesis. These results indicate that dietary copper deficiency, and the resultant decrease in SOD activity, depresses aortic PGI2 synthesis.