Yves Rayssiguier

Effects of two fermentable carbohydrates (inulin and resistant starch) and their combination on calcium and magnesium balance in rats

Resistant starch and inulin are complex carbohydrates that are fermented by the microflora and known to increase colonic absorption of minerals in animals. The fermentation of these substrates in the large bowel to short-chain fatty acids is the main reason for this increase in mineral absorption. The purpose of the present study was to examine the potential synergistic effect of a combination of these two fermentable carbohydrates. For this purpose, thirty-two adult male Wistar rats weighing 200 g were used in the present study. The rats were distributed into four groups, and fed for 21 d a fibre-free basal purified diet or diet containing 100 g inulin, or 150 g resistant starch (raw potato starch)/kg diet or a blend of 50 g inulin and 75 g resistant starch/kg diet. After an adaptation period of 14 d, the rats were then transferred to metabolic cages and dietary intake, faeces and urine were monitored for 5 d. The animals were then anaesthetized and caecal Ca and Mg absorption were measured. Finally, the rats were killed and blood, caecum and tissues were sampled. Ca and Mg levels were assessed in diets, faeces, urine, caecum and plasma by atomic absorption spectrometry. Our results confirmed that inulin and resistant starch ingestion led to considerable caecal fermentation in the three experimental groups compared with the control group diet. Moreover, both carbohydrates significantly increased the intestinal absorption and balance of Ca and Mg, without altering the plasma level of these two minerals. Interestingly, the combination of the studied carbohydrates increased significantly the caecal soluble Ca and Mg concentrations, the apparent intestinal absorption and balance of Ca, and non-significantly the plasma Mg level. In conclusion, a combination of different carbohydrates showed synergistic effects on intestinal Ca absorption and balance in rats. Further studies with other types of carbohydrate combinations should be carried out to extend these findings.

Inflammatory response following acute magnesium deficiency in the rat

The importance of inflammatory processes in the pathology of Mg deficiency has been recently reconsidered but the sequence of events leading to the inflammatory response remains unclear. Thus, the purpose of the present study was to characterize more precisely the acute phase response following Mg deficiency in the rat. Weaning male Wistar rats were pair-fed either a Mg-deficient or a control diet for either 4 or 8 days. The characteristic allergy-like crisis of Mg-deficient rats was accompanied by a blood leukocyte response and changes in leukocytes subpopulations. A significant increase in interleukin-6 (IL-6) plasma level was observed in Mg-deficient rats compared to rats fed a control diet. The inflammatory process was accompanied by an increase in plasma levels of acute phase proteins. The concentrations of alpha2-macroglobulin and alpha1-acid glycoprotein in the plasma of Mg-deficient rats were higher than in control rats. This was accompanied in the liver by an increase in the level of mRNA coding for these proteins. Moreover, Mg-deficient rats showed a significant increase in plasma fibrinogen and a significant decrease in albumin concentrations. Macrophages found in greater number in the peritoneal cavity of Mg-deficient rats were activated endogenously and appeared to be primed for superoxide production following phorbol myristate acetate stimulation. A high plasma level of IL-6 could be detected as early as day 4 for the Mg-deficient diet. Substance P does not appear to be the initiator of inflammation since IL-6 increase was observed without plasma elevation of this neuropeptide. The fact that the inflammatory response was an early consequence of Mg deficiency suggests that reduced extracellular Mg might be responsible for the activated state of immune cells.

Five‐Week Intake of Short‐Chain Fructo‐Oligosaccharides Increases Intestinal Absorption and Status of Magnesium in Postmenopausal Women

Fermentable carbohydrates have been shown to be nondigestible by human enzymes in the small intestine but are fermented extensively in the large bowel to short‐chain fatty acids (SCFAs), which can increase mineral absorption. It has been shown that feeding such carbohydrates including short‐chain fructo‐oligosaccharides (sc‐FOSs) increases intestinal magnesium (Mg) absorption in animals, but their beneficial impact on Mg absorption in humans still remains to be established. Therefore, this work aimed to investigate the effect of moderate daily doses of sc‐FOSs (10 g/day) on the intestinal absorption and status of Mg in postmenopausal women without hormone replacement therapy (HRT). Eleven healthy postmenopausal women aged 59 ± 6 years (mean ± SD) received for 5 weeks sc‐FOS or sucrose (placebo) treatments according to a randomized, double‐blind, crossover design separated by a washout period of at least 3 weeks. Subjects ingested 87.5 mg of stable isotope25Mg together with a fecal marker. Subsequently, feces were collected for 5–7 days. An inductively coupled plasma mass spectrometer (ICP/MS) was used for25Mg stable isotope measurements in feces, urine, and blood. Mg levels were assessed also at the beginning and at the end of each treatment in plasma, erythrocytes, and urine. These measurements allowed for the determination of net intestinal Mg absorption and Mg status. The results show that the addition of 10 g sc‐FOS to the diet increased Mg absorption by 12.3%, from 30.2 ± 5.0% (placebo treatment) to 33.9 ± 7.2% (sc‐FOS treatment; mean ± SD; p < 0.02). This increase in intestinal Mg absorption was accompanied by an increase in plasma25Mg level and led to a higher urinary25Mg excretion. This is the first time that such an effect is shown in humans. The overall conclusion of this work is that the ingestion of moderate doses of sc‐FOS did improve intestinal Mg absorption and status in postmenopausal women. Because of the important role of Mg in many cellular functions, such Mg absorption improvement may be particularly interesting when the dietary intake of Mg is limited.

Plasma levels of 8-epiPGF2α, an in vivo marker of oxidative stress, are not affected by aging or Alzheimer’s disease

Free radicals are likely involved in the aging process and there is a growing body of evidence that free radical damage to cellular function is associated with a number of age-related diseases such as atherosclerosis, cancer, and neurologic disorders. The present study was designed to evaluate in a healthy population the evolution with age of 8-epiPGF2alpha plasma levels, a recently proposed marker of in vivo lipid peroxidation. Moreover we investigated this marker of oxidative stress in patients with Alzheimers disease (AD), an age-related neurodegenerative disorder in the development of which free radicals have been involved. Our results show that in the healthy population studied, despite decreased antioxidant defenses with increasing age as monitored by antioxidant capacity measurement, plasma 8-epiPGF2alpha levels were not correlated with age. Moreover, we have demonstrated that AD patients presented no modification of plasma 8-epiPGF2alpha level and no major alteration of the antioxidant status. In conclusion, the measurement of plasma 8-epiPGF2alpha did not allow us to detect alterations in oxidative stress with aging or in AD.

Dietary magnesium affects susceptibility of lipoproteins and tissues to peroxidation in rats.

Magnesium (Mg)-deficient and control diets were pair-fed to weanling Wistar rats for 8 days. Plasma lipoproteins were separated into various density classes by sequential preparative ultracentrifugation. The extent of lipid peroxidation was measured in terms of thiobarbituric acid reactive substances in lipoproteins and tissue homogenates before or after iron-induced lipid peroxidation. Hyperlipemia in Mg-deficient rats was accompanied by increased oxidation of very-low-density lipoproteins and low-density lipoproteins. Moreover, very-low-density lipoproteins and high-density lipoproteins from Mg-deficient rats were more susceptible to oxidative damage following iron incubation. Mg deficiency increased lipid peroxidation in liver, heart and skeletal muscles. Their homogenates were more susceptible to in vitro peroxidation. Mg deficiency has been discussed as a possible contributory factor in the development of cardiovascular disease and was associated with tissue damage and membrane alteration. These results demonstrate for the first time that Mg affects the susceptibility of lipoproteins to peroxidation and suggest that the mechanism responsible for the pathological consequences of Mg deficiency may be mediated by lipid peroxidation products.

Increased phagocytosis and production of reactive oxygen species by neutrophils during magnesium deficiency in rats and inhibition by high magnesium concentration.

Recent studies underline the importance of the immunoinflammatory processes in the pathology of Mg deficiency. Neutrophils possess a superoxide anion-generating NADPH oxidase and its inappropriate activation may result in tissue damage. The aim of the present study was to assess the effect of experimental Mg deficiency in the rat on polymorphonuclear leucocytes (PMN) activity and the role of increasing extracellular Mg. Weaning male Wistar rats were fed either a Mg-deficient or a control diet for 8 d. In Mg-deficient rats, the characteristic inflammatory response was accompanied by a marked increase in the number of PMN. Higher plasma interleukin 6 and NO concentrations and increased lipid peroxidation in the heart were found in Mg-deficient rats as compared with control rats. As shown by chemiluminescence studies, basal neutrophil activity from Mg-deficient rats was significantly elevated when compared with neutrophils from control rats. Moreover, the chemiluminescence of PMN from Mg-deficient rats was significantly higher than that of control rats following phorbol myristate acetate or opsonized zymosan activation. PMN from Mg-deficient rats also showed an increased activity of phagocytosis in comparison with neutrophils from control animals. Increasing extracellular Mg concentration in the incubating medium of PMN (0.8 v. 8.0 mM) decreased the chemiluminescence activity of PMN from control rats following opsonized zymosan activation. Chemiluminescence activities of PMN from Mg-deficient rats following phorbol myristate acetate or opsonized zymosan challenge were also decreased by high extracellular Mg concentration. From this work, it appears that PMN activation is an early consequence of Mg deficiency and that high extracellular Mg concentration inhibits free radicals generation.

Role of spinal NMDA receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced by magnesium deficiency in rats

Magnesium (Mg)‐deficient rats develop a mechanical hyperalgesia which is reversed by a N‐Methyl‐D‐Aspartate (NMDA) receptor antagonist. Given that functioning of this receptor‐channel is modulated by Mg, we wondered whether facilitated activation of NMDA receptors in Mg deficiency state may in turn trigger a cascade of specific intracellular events present in persistent pain. Hence, we tested several antagonists of NMDA and non‐NMDA receptors as well as compounds interfering with the functioning of intracellular second messengers for effects on hyperalgesia in Mg‐deficient rats. Hyperalgesic Mg‐deficient rats were administered intrathecally (10 μl) or intraperitoneally with different antagonists. After drug injection, pain sensitivity was evaluated by assessing the vocalization threshold in response to a mechanical stimulus (paw pressure test) over 2 h. Intrathecal administration of MgSO4 (1.6, 3.2, 4.8, 6.6 μmol) as well as NMDA receptor antagonists such as MK‐801 (0.6, 6.0, 60 nmol), AP‐5 (10.2, 40.6, 162.3 nmol) and DCKA (0.97, 9.7, 97 nmol) dose‐dependently reversed the hyperalgesia. Chelerythrine chloride, a protein kinase C (PKC) inhibitor (1, 10.4, 104.2 nmol) and 7‐NI, a specific nitric oxide (NO) synthase inhibitor (37.5, 75, 150 μmol kg−1, i.p.) induced an anti‐hyperalgesic effect in a dose‐dependent manner. SR‐140333 (0.15, 1.5, 15 nmol) and SR‐48968 (0.17, 1.7, 17 nmol), antagonists of neurokinin receptors, produced a significant, but moderate, increase in vocalization threshold. These results demonstrate that Mg‐deficiency induces a sensitization of nociceptive pathways in the spinal cord which involves NMDA and non‐NMDA receptors. Furthermore, the data is consistent with an active role of PKC, NO and, to a lesser extent substance P in the intracellular mechanisms leading to hyperalgesia.

Accelerated thymus involution in magnesium-deficient rats is related to enhanced apoptosis and sensitivity to oxidative stress

Experimental Mg deficiency leads to alterations in the immune response. Reduction of thymus weight and histological changes were previously observed in Mg-deficient rats after several weeks on a deficient diet, suggesting that functions of this immune organ may be affected by Mg deficiency. More recently, changes in the immune system during early Mg deficiency were shown. Thus, in the present study we examined modifications in the thymus during the early stages of Mg deficiency in weanling rats. From our results, it appears that Mg deficiency accelerates thymus involution. The assessment of apoptosis (enumeration of apoptotic cells on the basis of morphological criteria and intranucleosomal degradation of genomic DNA) showed greater values in thymuses from Mg-deficient rats as compared with controls. This was observed very early, since a significant difference was shown on the second day of deficiency, before reduced weight of thymus, which was recorded in the later period. These results indicate the relationship of accelerated thymus involution with an active process of cell death. Mg deficiency led to histological changes in the thymus. In the early stage of deficiency (second day) the presence of inflammatory cells was shown, suggesting that the inflammatory process was already occurring in the tissue studied. Later (eighth day) an increased proportion of epithelial reticular cells in the cortex was shown, indicating a remodelling process occurring in this period. Enhanced susceptibility to peroxidation also occurred very early during Mg deficiency. It may be hypothesized that disturbances in Mg status of short duration could have cellular effects with various deleterious consequences.

Rats fed a high sucrose diet have altered heart antioxidant enzyme activity and gene expression

Several studies in human and animal models have shown that consumption of fructose facilitates oxidative damage but the mechanisms involved are unclear. In this study, the effects of two weeks of high sucrose on both oxidative stress parameters and stress-related gene expression, using a cDNA array, were investigated in rat heart. Both increased TBARS and lower Cu-Zn-SOD activity were found in heart from high sucrose fed rats compared to rats on a starch diet. Higher plasma NO level was also found in the high sucrose group, corroborating the pro-oxidant effect of fructose. The Cu-Zn-SOD mRNA level was also greater in the high sucrose group; the Mn-SOD, GPX and catalase were not different between the two groups. Increased HSP70 and decreased COMT genes expression were observed, underlying the hypertensive effect of dietary fructose. These findings confirm the pro-oxidant effect of high sucrose feeding to rats and highlight the NO/O(2)(*-) balance importance in oxidative homeostasis.

Enhanced tumor necrosis factor-α production following endotoxin challenge in rats is an early event during magnesium deficiency

Magnesium (Mg) plays an essential role in fundamental cellular reactions and the importance of the immuno-inflammatory processes in the pathology of Mg deficiency has been recently reconsidered. The purpose of the present study was to assess the effect of different stages of Mg deficiency on endotoxin response and tumor necrosis factor-alpha (TNF alpha) production. Weaning male Wistar rats were pair fed either a Mg-deficient or a control diet. At day 7, lipopolysaccharide (LPS) induced no lethal effects in control rats but resulted in 70% mortality in Mg-deficient rats within 3 h. The vulnerability of Mg-deficient rats to LPS was associated with higher TNF alpha plasma values. Mg-deficient animals that received magnesium supplementation before endotoxin challenge had significantly increased survival. At day 2, control and Mg-deficient rats were also subjected to endotoxin challenge with or without magnesium pre-treatment. A significant increase in TNF alpha plasma level was observed in Mg-deficient rats compared to rats fed the control diet. Mg-deficient rats that received magnesium replacement therapy before endotoxin challenge had significantly lower TNF alpha plasma values than those receiving saline before endotoxin. Thus, the results of this experiment suggest that the activated or primed state of immune cells is an early event occurring in Mg deficiency.