Inositols: From Established Knowledge to Novel Approaches.

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

Inositol Hexaphosphate (IP6) and Colon Cancer: From Concepts and First Experiments to Clinical Application.

Multiple human health-beneficial effects have been related to highly phosphorylated inositol hexaphosphate (IP6). This naturally occurring carbohydrate and its parent compound, myo-inositol (Ins), are abundantly present in plants, particularly in certain high-fiber diets, but also in mammalian cells, where they regulate important cellular functions. However, the striking and broad-spectrum anticancer activity of IP6, consistently demonstrated in different experimental models, has been in a spotlight of the scientific community dealing with the nutrition and cancer during the last several decades. First experiments were performed in colon cancer 30 years ago. Since then, it has been shown that IP6 reduces cell proliferation, induces apoptosis and differentiation of malignant cells with reversion to normal phenotype, affecting several critical molecular targets. Enhanced immunity and antioxidant properties also contribute to the tumor cell destruction. Although Ins possesses a modest anticancer potential, the best anticancer results were obtained from the combination of IP6 + Ins. Here we review the first experimental steps in colon cancer, when concepts and hypotheses were put together almost without real knowledge and present clinical studies, that were initiated in colon cancer patients. Available as a dietary supplement, IP6 + Ins has been shown to enhance the anticancer effect of conventional chemotherapy, controls cancer metastases, and improves quality of life in cancer patients. Emerging clinical and still vast amount of experimental data suggest its role either as an adjuvant or as an "alternative" to current chemotherapy for cancer.

Role of Inositols and Inositol Phosphates in Energy Metabolism.

Recently, inositols, especially myo-inositol and inositol hexakisphosphate, also known as phytic acid or IP6, with their biological activities received much attention for their role in multiple health beneficial effects. Although their roles in cancer treatment and prevention have been extensively reported, interestingly, they may also have distinctive properties in energy metabolism and metabolic disorders. We review inositols and inositol phosphate metabolism in mammalian cells to establish their biological activities and highlight their potential roles in energy metabolism. These molecules are known to decrease insulin resistance, increase insulin sensitivity, and have diverse properties with importance from cell signaling to metabolism. Evidence showed that inositol phosphates might enhance the browning of white adipocytes and directly improve insulin sensitivity through adipocytes. In addition, inositol pyrophosphates containing high-energy phosphate bonds are considered in increasing cellular energetics. Despite all recent advances, many aspects of the bioactivity of inositol phosphates are still not clear, especially their effects on insulin resistance and alteration of metabolism, so more research is needed.

Anticancer Properties of Inositol Hexaphosphate and Inositol: An Overview.

Inositol hexaphosphate (IP6) and its parent compound myo-inositol (Ins) are active compounds from rice and other grains, with a broad spectrum of biological activities important in health and diseases. However, the most striking is the anticancer effect of IP6 and Ins that has been actively investigated during the last decades. A consistent and reproducible anticancer action of IP6 has been demonstrated in various experimental models. IP6 reduces cell proliferation, induces apoptosis and differentiation of malignant cells via PI3K, MAPK, PKC, AP-1 and NF-kappaB. Very few clinical studies in humans and case reports have indicated that IP6 is able to enhance the anticancer effect of conventional chemotherapy, control cancer metastases, and improve quality of life. Reduced burden of chemotherapy side-effects in patients receiving IP6 alone or in combination with Ins has been reported. Because of the highly promising preclinical and emerging clinical data, large clinical trials and further mechanistic studies are warranted.

SIRT3 regulates cancer cell proliferation through deacetylation of PYCR1 in proline metabolism.

SIRT3 is a major mitochondrial deacetylase, which regulates various metabolic pathways by deacetylation; however, the effect of SIRT3 on proline metabolism is not reported. Pyrroline-5-carboxylate reductase 1 (PYCR1) participates in proline synthesis process by catalyzing the reduction of P5C to proline with concomitant generation of NAD+ and NADP+. PYCR1 is highly expressed in various cancers, and it can promote the growth of tumor cells. Here, through immunoprecipitation and mass spectrometry, we found that PYCR1 is in SIRT3's interacting network. PYCR1 directly binds to SIRT3 both in vivo and in vitro. CBP is the acetyltransferase for PYCR1, whereas SIRT3 deacetylates PYCR1. We further identified that K228 is the major acetylation site for PYCR1. Acetylation of PYCR1 at K228 reduced its enzymatic activity by impairing the formation of the decamer of PYCR1. As a result, acetylation of PYCR1 at K228 inhibits cell proliferation, while deacetylation of PYCR1 mediated by SIRT3 increases PYCR1's activity. Our findings on the regulation of PYCR1 linked proline metabolism with SIRT3, CBP and cell growth, thus providing a potential approach for cancer therapy.

Obesity and cancer risk: evidence, mechanisms, and recommendations.

Obesity, a growing health problem worldwide, has been associated with the metabolic syndrome, diabetes, cardiovascular disease, hypertension, and other chronic diseases. Recently, the obesity-cancer link has received much attention. Epidemiological studies have shown that obesity is also associated with increased risk of several cancer types, including colon, breast, endometrium, liver, kidney, esophagus, gastric, pancreatic, gallbladder, and leukemia, and can also lead to poorer treatment and increased cancer-related mortality. Biological mechanisms underlying the relationship between obesity and cancer are not well understood. They include modulation of energy balance and calorie restriction, growth factors, multiple signaling pathways, and inflammatory processes. Key among the signaling pathways linking obesity and cancer is the PI3K/Akt/mTOR cascade, which is a target of many of the obesity-associated factors and regulates cell proliferation and survival. Understanding the molecular and cellular mechanisms of the obesity-cancer connection is important in developing potential therapeutics. The link between obesity and cancer underscores the recommendation to maintain a healthy body weight throughout life as one of the most important ways to protect against cancer.

Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice.

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.

Pharmacokinetics and tissue distribution of inositol hexaphosphate in C.B17 SCID mice bearing human breast cancer xenografts.

Inositol hexaphosphate (IP(6)) is effective in preclinical cancer prevention and chemotherapy. In addition to cancer, IP(6) has many other beneficial effects for human health, such as reduction in risk of developing cardiovascular disease and diabetes and inhibition of kidney stone formation. Studies presented here describe the pharmacokinetics, tissue distribution, and metabolism of IP(6) following intravenous (IV) or per os (PO) administration to mice. SCID mice bearing MDA-MB-231 xenografts were treated with 20 mg/kg IP(6) (3 µCi per mouse [(14)C]-uniformly ring-labeled IP(6)) and euthanized at various times after IP(6) treatment. Plasma and tissues were analyzed for [(14)C]-IP(6) and metabolites by high-performance liquid chromatography with radioactivity detection. Following IV administration of IP(6), plasma IP(6) concentrations peaked at 5 minutes and were detectable until 45 minutes. Liver IP(6) concentrations were more than 10-fold higher than plasma concentrations, whereas other normal tissue concentrations were similar to plasma. Only inositol was detected in xenografts. After PO administration, IP(6) was detected in liver; but only inositol was detectable in other tissues. After both IV and PO administration, exogenous IP(6) was rapidly dephosphorylated to inositol; however, alterations in endogenous IPs were not examined.

Effect of inositol hexaphosphate on the development of UVB-induced skin tumors in SKH1 hairless mice.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is abundant in many plants and in various high-fiber foods, such as cereals and legumes. IP6 has a striking, broad-spectrum anticancer activity in various in vitro and animal models, in which it interferes with key pathways in malignancy to inhibit cell proliferation, cell-cycle progression, metastasis, invasion, and angiogenesis and to induce apoptosis. In this study, we investigated the protective effects of IP6 in drinking water on the incidence of UVB-induced skin cancer in the SKH1 (Crl: SKH1-hr) mouse model. One group of 15 mice received 2% IP6 in drinking water and UVB exposure, and the other group (n = 15) received UVB exposure only. All mice in both groups were fed an IP6-deficient diet (AIN 76A). The treatment group started receiving 2% IP6 in the drinking water 3 d before irradiation. Mice were irradiated 3 times each week, starting at a dose of 1.5 kJ/m2, with weekly increases in increments of 1.5 kJ/m2 to a final dose of 7.5 kJ/m2. Tumor formation was monitored until the week 31. IP6 in drinking water significantly decreased tumor incidence by 5-fold and tumor multiplicity by 4-fold. These results show that IP6 has an antiphotocarcinogenic effect and can protect against UVB-induced tumor formation.

Protection against cancer by dietary IP6 and inositol.

Inositol hexaphosphate (IP(6)) is a naturally occurring polyphosphorylated carbohydrate, abundantly present in many plant sources and in certain high-fiber diets, such as cereals and legumes. In addition to being found in plants, IP(6) is contained in almost all mammalian cells, although in much smaller amounts, where it is important in regulating vital cellular functions such as signal transduction, cell proliferation, and differentiation. For a long time IP(6) has been recognized as a natural antioxidant. Recently IP(6) has received much attention for its role in cancer prevention and control of experimental tumor growth, progression, and metastasis. In addition, IP(6) possesses other significant benefits for human health, such as the ability to enhance immune system, prevent pathological calcification and kidney stone formation, lower elevated serum cholesterol, and reduce pathological platelet activity. In this review we show the efficacy and discuss some of the molecular mechanisms that govern the action of this dietary agent. Exogenously administered IP(6) is rapidly taken up into cells and dephosphorylated to lower inositol phosphates, which further affect signal transduction pathways resulting in cell cycle arrest. A striking anticancer action of IP(6) was demonstrated in different experimental models. In addition to reducing cell proliferation, IP(6) also induces differentiation of malignant cells. Enhanced immunity and antioxidant properties also contribute to tumor cell destruction. Preliminary studies in humans show that IP(6) and inositol, the precursor molecule of IP(6), appear to enhance the anticancer effect of conventional chemotherapy, control cancer metastases, and improve quality of life. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP(6) + inositol holds great promise in our strategies for cancer prevention and therapy. There is clearly enough evidence to justify the initiation of full-scale clinical trials in humans.

Inositol hexaphosphate (IP6) blocks proliferation of human breast cancer cells through a PKCdelta-dependent increase in p27Kip1 and decrease in retinoblastoma protein (pRb) phosphorylation.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate with demonstrated anti-proliferative and anti-cancer activity in mammary cells. We hypothesized that IP6 modulates cell cycle proteins by action on cytoplasmic signaling molecules. The effects of both pharmacological (2 mM) and physiological (100 microM) doses of IP6 on major PKC isoforms (PKCalpha, delta, epsilon, beta and zeta), PI3-K/Akt and ras/Erk1/2 were evaluated. Treatment of MCF-7 human breast cancer cells with 2 mM IP6 for 24 h caused a 3.1-fold increase in the expression of anti-proliferative PKCdelta. Similar results were observed with 100 microM IP6 at only 30-60 min post-treatment. IP6 also caused an increase in PKCdelta activity, shown by its translocation from cytosol to membrane. No changes in expression of PKC alpha, delta, epsilon, beta and zeta were detected. Additionally, IP6 caused a decrease of Erk1/2 and Akt activity. Among cell cycle control proteins, IP6 resulted in increased p27Kip1 protein levels and marked reduction of pRb phosphorylation. Specificity of the IP6 effects on p27Kip1 and pRb in MCF-7 cells (hormone-dependent) were additionally confirmed in highly invasive hormone-independent MDA-MB 231 breast cancer cells. Use of specific pharmaclogical inhibitors of PKC delta, MEK/Erk, and PI3K/Akt pathways indicated that the IP6-mediated effects on PKC delta were responsible for up-regulation of p27Kip, and pRb hypo-phosphorylation. In addition, IP6-induced apoptosis detected in MCF-7 cells appeared also to be PKC delta-dependent. Our data suggest potential usefulness of IP6 as a novel therapeutic modulator of PKC delta and p27Kip1, an important prognostic factor in human breast cancers.

Anti-angiogenic activity of inositol hexaphosphate (IP6).

A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP(6)) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP(6) reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP(6) on both. IP(6) inhibited the proliferation and induced the differentiation of endothelial cells in vitro; the growth of bovine aortic endothelial cells (BAECs) evaluated by MTT proliferation assay was inhibited in a dose-dependent manner (IC(50) = 0.74 mM). The combination of IP(6) and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP(6) inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP(6) significantly reduced basic fibroblast growth factor (bFGF)-induced vessel formation (P < 0.01) in vivo in Matrigel plug assay. Exposure of HepG2, a human hepatoma cell line, to IP(6) for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RT-PCR. IP(6) treatment of HepG2 cells for 24 h also significantly reduced the VEGF protein levels in conditioned medium, in a concentration-dependent manner (P = 0.012). Thus, IP(6) has an inhibitory effect on induced angiogenesis.

Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: I. In vitro studies of adhesion, migration and invasion of MDA-MB 231 human breast cancer cells.

BACKGROUND: The anti-cancer agent inositol hexaphosphate (IP6) is an abundant intrinsic component of both plant and mammalian cells. In addition to inducing differentiation and inhibiting growth of numerous cancer cell lines in vitro, IP6 has been demonstrated to prevent and abrogate both primary tumor and metastasis in vivo. MATERIALS AND METHODS: Using MDA-MB 231 human breast cancer cells, we studied the potential of IP6 to inhibit cell adhesion, migration and invasion, the key steps in cancer metastasis, utilizing the extracellular matrix (ECM) proteins, a culture wounding assay, modified Boyden chambers, immunocytochemistry and zymography. RESULTS: IP6 treatment caused a 65% reduction of cell adhesion to fibronection (p = 0.002) and a 37% reduction to collagen (p = 0.005). To determine whether a decrease in cell adhesion leads to a decrease in cell motility, migration assays were performed; IP6 decreased both the number of migrating cells and the distance of cell migration into the denuded area by 72% (p < 0.001). Haptotatic cell migration in a modified Boyden chambers was also reduced in a dose-dependent manner. While cell migration on fibronectin was inhibited by 65% (p < 0.001), migration on collagen and laminin was decreased by 32% (p < 0.01) and 13% (p < 0.05), respectively. Immunocytochemistry revealed the absence of lamellipodia structure in IP6-treated cells as compared to untreated cells, corresponding to a diminished ability of cancer cells to form cellular network as determined by Matrigel outgrowth assay. Likewise, cell invasion also was decreased (by 72% after IP6 treatment, p = 0.001) in a dose-dependent fashion. Additionally, IP6 significantly (p = 0.006) inhibited the secretion of matrix metalloproteinase (MMP)-9 as assessed by zymography. CONCLUSION: The results of this study show that IP6 inhibits the metastasis of human breast cancer cells in vitro through effects on cancer cell adhesion, migration and invasion.

Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: II. Effects on integrins and focal adhesions.

BACKGROUND: We have shown that inositol hexaphosphate (IP6), a natural compound and a potent anti-cancer agent, inhibited cancer cell adhesion to the extracellular matrix (ECM) proteins, thereby leading to inhibition of cell migration and invasion. Cell adhesion to ECM is mediated by specific cell surface integrins, which transduce intracellular signals through their interaction and activation of other proteins that are recruited to the focal adhesion. We hypothesize that IP6 decreases cell adhesion by suppressing the integrin receptors and their subsequent signaling pathway. MATERIALS AND METHODS: We analyzed integrin expressions of the highly invasive estrogen receptor-negative human breast cancer MDA-MB 231 cells exposed to IP6 by flow cytometry. The expression of focal adhesion proteins was investigated by immunocytochemistry and Western blotting. RESULTS: IP6 treatment caused a significant (P < 0.005) decrease in the expression of integrin heterodimers alpha 2 beta 1 (collagen receptor), alpha 5 beta 1 (fibronectin receptor) and alpha v beta 3 (vitronectin receptor); flow cytometry showed that it was the alpha 5 subunit that was down-regulated ( < 0.001). However, the expression of the alpha 2, alpha v, beta 1 and beta 3 subunits were not affected by IP6 treatment. When the expression of integrins on the cell surface was assessed, there was a dramatic 82% decrease in the expression of alpha 5 beta 1 on IP6-treated cells (P < 0.0001), indicating a decrease in cell surface expression of the heterodimers. No effect was seen when inositol hexasulfate (IS6), an analogue of IP6, was used as a control. Immunocytochemistry showed a lack of clustering of paxillin; tyrosine-phosphorylated proteins in IP6-treated cells were discontinuous and scattered around the cell periphery, whereas the patterns were more dense and localized in control cells. Consistent with these observations, focal adhesion kinase (FAK) autophosphorylation at tyrosine-397 residue was suppressed, albeit modestly, by IP6 treatment, suggesting a down-regulation in the integrin-mediated signaling pathway. CONCLUSION: The results of this study indicate that IP6-induced inhibition of cancer cell adhesion, migration and invasion may be mediated through the modulation of integrin dimerization, cell surface expression and integrin-associated signaling pathway.

Cancer inhibition by inositol hexaphosphate (IP6) and inositol: from laboratory to clinic.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is present in substantial amounts in almost all plant and mammalian cells. It was recently recognized to possess multiple biological functions. A striking anticancer effect of IP6 was demonstrated in different experimental models. Inositol is also a natural constituent possessing moderate anticancer activity. The most consistent and best anticancer results were obtained from the combination of IP6 plus inositol. In addition to reducing cell proliferation, IP6 increases differentiation of malignant cells, often resulting in a reversion to normal phenotype. Exogenously administered IP6 is rapidly taken into the cells and dephosphorylated to lower-phosphate inositol phosphates, which further interfere with signal transduction pathways and cell cycle arrest. Enhanced immunity and antioxidant properties can also contribute to tumor cell destruction. However, the molecular mechanisms underlying this anticancer action are not fully understood. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP6 holds great promise in our strategies for the prevention and treatment of cancer. IP6 plus inositol enhances the anticancer effect of conventional chemotherapy, controls cancer metastases, and improves the quality of life, as shown in a pilot clinical trial. The data strongly argue for the use of IP6 plus inositol in our strategies for cancer prevention and treatment. However, the effectiveness and safety of IP6 plus inositol at therapeutic doses needs to be determined in phase I and phase II clinical trials in humans.

Inositol hexaphosphate (IP6) enhances the anti-proliferative effects of adriamycin and tamoxifen in breast cancer.

The current treatment of breast carcinomas recognizes the importance of combination therapy in order to increase efficacy and decrease side effects of conventional chemotherapy. Inositol hexaphosphate (IP6), a naturally occurring polyphosphorylated carbohydrate, has shown a significant anti-cancer effect in various in vivo and in vitro models, including breast cancer. In this study, we investigated the in vitro growth inhibitory activity of IP6 in combination with adriamycin or tamoxifen, against three human breast cancer cell lines: estrogen receptor (ER) alpha-positive MCF-7, ER alpha-negative MDA-MB 231 and adriamycin-resistant MCF-7 (MCF-7/Adr) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Much lower concentrations of IP6 were required after 96 h of treatment to inhibit the growth of MCF-7/Adr cells than MCF-7 cells; the IC50 for MCF-7/Adr cells was 1.26 mM compared to 4.18 mM for MCF-7 cells. The ER-negative MDA-MB 231 cells were also highly sensitive to IP6 with IC50 being 1.32 mM. To determine the effects of IP6 in combination with either adriamycin or tamoxifen, the median effect principle and Webb's fraction method were used to determine the combination index (CI) and the statistical differences. Growth suppression was markedly increased when IP6 was administered prior to the addition of adriamycin, especially against MCF-7 cells (CI = 0.175 and p < 0.0001). Synergism was also observed when IP6 was administered after tamoxifen in all three cell lines studied (CI = 0.343, 0.701 and 0.819; p < 0.0001, p = 0.0003 and 0.0241 for MCF-7/Adr, MCF-7 and MDA-MB 231, respectively). The growth of primary culture of breast cancer cells from patients was inhibited by IP6 with LC50 values ranging from 0.91 to 5.75 mM (n = 10). Our data not only confirm that IP6 alone inhibits the growth of breast cancer cells; but it also acts synergistically with adriamycin or tamoxifen, being particularly effective against ER alpha-negative cells and adriamycin-resistant cell lines.

Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells, tissues and biological fluids.

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.