Alexander J. Michels

Grape seed and tea extracts and catechin 3-gallates are potent inhibitors of α-amylase and α-glucosidase activity.

This study evaluated the inhibitory effects of plant-based extracts (grape seed, green tea, and white tea) and their constituent flavan-3-ol monomers (catechins) on α-amylase and α-glucosidase activity, two key glucosidases required for starch digestion in humans. To evaluate the relative potency of extracts and catechins, their concentrations required for 50 and 90% inhibition of enzyme activity were determined and compared to the widely used pharmacological glucosidase inhibitor, acarbose. Maximum enzyme inhibition was used to assess relative inhibitory efficacy. Results showed that grape seed extract strongly inhibited both α-amylase and α-glucosidase activity, with equal and much higher potency, respectively, than acarbose. Whereas tea extracts and catechin 3-gallates were less effective inhibitors of α-amylase, they were potent inhibitors of α-glucosidase. Nongallated catechins were ineffective. The data show that plant extracts containing catechin 3-gallates, in particular epigallocatechin gallate, are potent inhibitors of α-glucosidase activity and suggest that procyanidins in grape seed extract strongly inhibit α-amylase activity.

Age-related decline of sodium-dependent ascorbic acid transport in isolated rat hepatocytes

This study investigated whether the age-related decline in hepatic ascorbic acid (AA) levels in rats was due to altered AA uptake. AA concentrations were 68% lower in freshly isolated hepatocytes from old (24-26 months) versus young (3-5 months; p<0.0005) Fischer 344 rats. When incubated with 100 microM AA, cells from old as compared to young rats showed a 66% decline in both the rate of AA transport and the steady state intracellular levels. Sodium-free media significantly reduced AA uptake, suggesting that the sodium-dependent vitamin C transporter (SVCT) was largely responsible for declines in AA transport. Analysis of SVCT messenger RNA (mRNA) levels shows that one isoform of this transport protein, SVCT1, declines 45% with age, with no significant changes in SVCT2 mRNA levels. These results show for the first time that sodium-dependent AA transport declines during the aging process, which may account for much of the loss in tissue AA content.

Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response.

Little is known about either the basal or stimulated homeostatic mechanisms regulating nuclear tenure of Nf-e2-related factor 2 (Nrf2), a transcription factor that mediates expression of over 200 detoxification genes. Our data show that stress-induced nuclear Nrf2 accumulation is largely from de novo protein synthesis, rather than translocation from a pre-existing cytoplasmic pool. HepG2 cells were used to monitor nuclear Nrf2 24h following treatment with the dithiol micronutrient (R)-α-lipoic acid (LA; 50μM), or vehicle. LA caused a ≥2.5-fold increase in nuclear Nrf2 within 1h. However, pretreating cells with cycloheximide (50μg/ml) inhibited LA-induced Nrf2 nuclear accumulation by 94%. Providing cells with the mTOR inhibitor, rapamycin, decreased basal Nrf2 levels by 84% after 4h, but LA overcame this inhibition. LA-mediated de novo protein translation was confirmed using HepG2 cells transfected with a bicistronic construct containing an internal ribosome entry sequence (IRES) for Nrf2, with significant (P<0.05) increase in IRES use under LA treatment. These results suggest that a dithiol stimulus mediates Nrf2 nuclear tenure via cap-independent protein translation. Thus, translational control of Nrf2 synthesis, rather than reliance solely on pre-existing protein, may mediate the rapid burst of Nrf2 nuclear accumulation following stress stimuli.

Human Genetic Variation Influences Vitamin C Homeostasis by Altering Vitamin C Transport and Antioxidant Enzyme Function

New evidence for the regulation of vitamin C homeostasis has emerged from several studies of human genetic variation. Polymorphisms in the genes encoding sodium-dependent vitamin C transport proteins are strongly associated with plasma ascorbate levels and likely impact tissue cellular vitamin C status. Furthermore, genetic variants of proteins that suppress oxidative stress or detoxify oxidatively damaged biomolecules, i.e., haptoglobin, glutathione-S-transferases, and possibly manganese superoxide dismutase, affect ascorbate levels in the human body. There also is limited evidence for a role of glucose transport proteins. In this review, we examine the extent of the variation in these genes, their impact on vitamin C status, and their potential role in altering chronic disease risk. We conclude that future epidemiological studies should take into account genetic variation in order to successfully determine the role of vitamin C nutriture or supplementation in human vitamin C status and chronic disease risk.

(R)-α-Lipoic Acid Treatment Restores Ceramide Balance in Aging Rat Cardiac Mitochondria

Inflammation results in heightened mitochondrial ceramide levels, which cause electron transport chain dysfunction, elevates reactive oxygen species, and increases apoptosis. As mitochondria in aged hearts also display many of these characteristics, we hypothesized that mitochondrial decay stems partly from an age-related ceramidosis that heretofore has not been recognized for the heart. Intact mitochondria or their purified inner membranes (IMM) were isolated from young (4-6 mo) and old (26-28 mo) rats and analyzed for ceramides by LC-MS/MS. Results showed that ceramide levels increased by 32% with age and three ceramide isoforms, found primarily in the IMM (e.g. C(16)-, C(18)-, and C(24:1)-ceramide), caused this increase. The ceramidosis may stem from enhanced hydrolysis of sphingomyelin, as neutral sphingomyelinase (nSMase) activity doubled with age but with no attendant change in ceramidase activity. Because (R)-α-lipoic acid (LA) improves many parameters of cardiac mitochondrial decay in aging and lowers ceramide levels in vascular endothelial cells, we hypothesized that LA may limit cardiac ceramidosis and thereby improve mitochondrial function. Feeding LA [0.2%, w/w] to old rats for two weeks prior to mitochondrial isolation reversed the age-associated decline in glutathione levels and concomitantly improved Complex IV activity. This improvement was associated with lower nSMase activity and a remediation in mitochondrial ceramide levels. In summary, LA treatment lowers ceramide levels to that seen in young rat heart mitochondria and restores Complex IV activity which otherwise declines with age.

A new twist on an old vitamin: human polymorphisms in the gene encoding the sodium-dependent vitamin C transporter 1.

Encoded by the gene SLC23A1, the sodium-dependent vitamin C transporter 1 (SVCT1) plays a critical role in hepatic portal absorption and renal reabsorption of vitamin C (ascorbic acid). Through these functions, SVCT1 controls plasma vitamin C homeostasis, as recently shown in SVCT1 knockout mice (1). Despite massive loss of ascorbic acid from the circulation by urinary excretion, these mice survive due to increased endogenous vitamin C synthesis. An argument can therefore be made that factors altering SVCT1 activity may critically affect vitamin C homeostasis in humans, one of several mammalian species lacking ascorbic acid synthesis. In this issue of the Journal, Timpson et al (2) report on a large cohort of .15,000 individuals combined from 5 independent observational studies in the United Kingdom to provide the first evidence that the SLC23A1 genotype affects plasma vitamin C status in humans. One of the 4 SLC23A1 single nucleotide polymorphisms (SNPs) examined, rs33972313, was associated with a highly significant reduction in plasma vitamin C concentrations (–5.98 lmol/L per modified allele). Remarkably, this effect was observed without adjustment for variables known to affect vitamin C status, such as dietary intake or cigarette smoking, and despite diverse study designs and differing methods used for plasma ascorbic acid analysis. These data, therefore, suggest that the rs33972313 SNP is a ‘‘nonconfounded proxy for variation in L-ascorbic acid at the population level’’ (2) that needs to be considered—potentially with other SLC23A1 genetic variants— in the design of future observational studies and randomized controlled trials investigating the role of vitamin C in humans. A central question borne of this research is whether rs33972313 and other SLC23A1 variants modify chronic disease risk. Prior studies have found no correlation of SNPs in SLC23A1 with gastric cancer (3) or advanced colorectal cancer (4). One study did report a correlation of 2 SLC23A1 SNPs with decreased risk of lymphoma (5); one of these SNPs, rs6596473, was associated with a significant reduction in follicular lymphoma and is now shown by Timpson et al (2) to modestly but significantly increase plasma ascorbic acid concentrations (12.86 lmol/L per modified allele in the British Women’s Heart and Health Study cohort). However, a limitation of these previous genetic studies (3–5) is that plasma vitamin C concentrations were not measured concurrently, and hence correlations with SNPs or disease risk could not be assessed. Nevertheless, the notion that vitamin C affects chronic disease risk is supported by many epidemiologic studies, including those investigated by Timpson et al (2). For example, in the European Prospective Investigation into Cancer and Nutrition Norfolk Study, a 20-lmol/L increase in plasma ascorbic acid was correlated with a highly significant 20% decrease in relative risk of all-cause mortality, and similar inverse associations were observed for cardiovascular disease and ischemic heart disease (6). The British Regional Heart Study supported these findings by showing a significant decrease in markers of inflammation and endothelial dysfunction in relation to high plasma vitamin C concentrations (7). Furthermore, there is extensive evidence from randomized controlled trials that vitamin C supplementation restores normal endothelial function and vasodilation in individuals with coronary risk factors or established coronary artery disease (8). Because the determinants of plasma vitamin C concentrations are multifactorial in nature, the effects of SLC23A1 genetic polymorphisms must be viewed in the context of diet and lifestyle factors known to influence vitamin C concentrations and disease risk. Although not the focus of their work, the data presented by Timpson et al (2) confirm that lower socioeconomic status, excessive alcohol consumption, and cigarette smoking are associated with significantly lower plasma vitamin C concentrations (2). As indicated above, the effects of the genetic variants studied were independent of all of these potential confounding factors (2), which underscores the pervasive nature of the rs33972313 genotype. Combining deleterious diet and lifestyle factors with SLC23A1 SNPs that negatively (rs33972313) or positively (rs6596473) affect plasma ascorbic acid status may amplify or reduce, respectively, an individual’s risk of developing vitamin C deficiency and adverse health outcomes. Conversely, the limitations that a deleterious SLC23A1 gene variant places on plasma vitamin C status may be overcome by dietary means. If SVCT1-independent routes exist for intestinal vitamin C absorption, as suggested by current research in SVCT1 knockout mice (1), then increasing dietary consumption