The present invention provides uses of multi-nutrient supplements to rescue aberrant biochemical pathways and reduce birth defect caused by maternal diabetes. Choice of supplements is based on the ability of each supplement to correct the following hyperglycemia-associated abnormalities: increased reactive oxygen species generation, abnormal membrane phospholipid metabolism, and decreased glutathione synthesis.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the study of congenital malformation. More specifically, the present invention relates to methods of preventing diabetes-related congenital malformation.

2. Description of the Related Art

Each year in the United States, about 150,000 babies--3% of all live births are born with a major congenital malformation. This problem is worse in offspring of women who have type 1 or 2 diabetes; 6%-10% of these babies are born with a major congenital malformation. Based on the National Health and Nutrition Examination Survey conducted during 1988-1994, 1.1% of women 20-39 years of age have type 1 or 2 diabetes, and the incidence of diabetes among women of childbearing age has been increasing over the past four decades. It is projected that the number of women of childbearing age with type 2 diabetes will double by 2010, suggesting that approximately 8,000 babies will be born each year in the United States with a congenital malformation secondary to type 1 or 2 diabetes.

Human observational studies have demonstrated a strong link between the extent of a mother's glycemic control and the incidence of congenital malformations in her offspring. The putative teratogenic effects of hyperglycemia are supported by studies that demonstrate a reduction in the incidence of birth defects following clinical intervention targeted at achieving euglycemia. When euglycemia is successfully maintained periconceptionally and during the first trimester, the prevalence of malformations is reduced to a level comparable to that of the general population. However, even with excellent compliance and clinical care, euglycemia may be difficult to achieve and maintain. In addition, it is possible that organogenesis can be affected by short periods of hyperglycemia that are not reflected in the averaged values of glycosylated hemoglobin levels, which are used to monitor glucose levels. A further obstacle is that most women with diabetes do not seek preconceptional care and most have unplanned pregnancies. Hence, a very important goal for public health is to develop and implement accessible intervention strategies to diminish the occurrence of these anomalies.

Both clinical cases and animal studies have clearly demonstrated that the main characteristics of maternal hyperglycemia-associated defects are organogenesis and underdevelopment. The organ systems most commonly affected include the central nervous, cardiovascular, gastrointestinal, craniofacial, genitourinary, and skeletal systems. Because the neural folds and heart develop early during embryogenesis, a higher incidence of malformations is often seen in these organs. In the central nervous system, abnormalities can be categorized as underdevelopment of the midbrain and hindbrain and failure of the neural tube to close at both anterior (rostral) and posterior (caudal) ends of the neural axis. The failure of posterior neural tube closure results in spina bifida, a common birth defect seen in newborns.

Convincing evidence from clinical and experimental studies demonstrates that diabetes-related hyperglycemia leads to sustained generation of reactive oxygen species (ROS) and depletion of antioxidant defense, resulting in intracellular oxidative stress due to an imbalance in intracellular reduction-oxidation (redox) homeostasis. Under normal physiological conditions, oxygen free radicals, including hydroxyl radicals, superoxide anions, singlet oxygen, and hydrogen peroxide (H.sub.2O.sub.2), are produced during cellular energy metabolism in sub cellular organelles such as mitochondria. The reactive oxygen species mediate intracellular signal transduction that regulates a wide range of cell functions, including proliferation, differentiation, and migration. However, under pathological conditions, excess reactive oxygen species can oxidize proteins, lipids, and DNA, causing cell injury and even cell death.

Over the past two decades, multiple clinical and experimental studies have evaluated the efficacy of maternal dietary supplements to decrease the rate of diabetic embryopathy. Supplements that potentially alter the underlying hyperglycemia-induced increases in oxidative stress, decreases in antioxidant defense, and alterations in membrane lipid metabolism are most relevant.

Lipoic acid and vitamin C reduce reactive oxygen species-mediated effects and support generation of other antioxidants. Lipoic acid is a naturally occurring antioxidant that is an effective scavenger of free radicals. In experimental models, lipoic acid has been shown to reduce malformation rate in diabetic pregnancies from 25% to 10%. In clinical studies of non-pregnant diabetic individuals, lipoic acid lowered the plasma lipid hydroperoxides, demonstrating a decrease in oxidative stress. This effect was maintained even in patients with high glucose levels. Vitamin C is a hydrophilic molecule that can scavenge several free radicals, including the hydroxyl radical. Experimental studies have demonstrated that vitamin C supplementation reduces the rate of embryonic malformations and embryo resorption.

Dietary supplements containing arachidonic acid (AA) and vitamin E prevent membrane lipid peroxidation and may be beneficial for primary prevention of diabetic embryopathy. In vitro and in vivo experimental studies demonstrated that maternal supplementation with arachidonic acid reduced hyperglycemia-induced aberrant membrane lipid metabolism, suggesting a protective effect. Vitamin E is a major lipid-soluble antioxidant that protects biological membranes from lipid peroxidation. Following a diet supplemented with vitamin E, pregnant diabetic rats have higher serum vitamin E levels and lower embryonic malformation and resorption rates than non-supplemented diabetic rats.

Both N-acetylcysteine (NAC), a cysteine progenitor, and folic acid, a methionine/cysteine precursor, increase glutathione synthesis. In experimental models, supplementation with N-acetylcysteine reduced development of peripheral neuropathy and embryopathy in diabetic rats. This may be due to increase in glutathione synthesis and antioxidant capacity in the cell. It is well accepted that dietary supplementation with folic acid prevents neural tube defects in both experimental and clinical models. In diabetic rats, folic acid levels are especially low in the heart, brain, kidney, and muscle.

For each supplement discussed, lipoic acid, vitamin C, arachidonic acid, vitamin E, N-acetylcysteine, and folic acid, animal and human studies have demonstrated that each nutrient is lower among diabetic individuals than non-diabetic individuals. Clinical trials among diabetic patients have demonstrated that a reduction in diabetic complications (i.e., glycemic control, neuropathy, nephropathy, retinopathy, vasculopathy) may occur following supplementation with these compounds. Most of the published studies, however, focused on the effects of a single nutrient, and no randomized clinical trials evaluating adjunctive therapy with antioxidants to prevent diabetic embryopathy have been published.

Thus, it is desirable to define an optimal intervention strategy of using combinations of nutrients for primary prevention of maternal hyperglycemia and diabetic embryopathy. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

In one embodiment, there are provided a dietary supplement and a method of using such supplement to reduce birth defect caused by maternal diabetes. The dietary supplement comprises lipoic acid, vitamin C, vitamin E, arachidonic acid, folic acid, and glutathione precursor N-acetylcysteine.

In another embodiment, there are provided a dietary supplement and a method of using such supplement to reduce birth defect caused by maternal diabetes. The dietary supplement comprises one or more antioxidants, one or more compounds that improve membrane lipid metabolism (e.g. arachidonic acid), and one or more compounds that support glutathione synthesis (e.g. methionine/cysteine precursor and glutathione precursor).

DETAILED DESCRIPTION OF THE INVENTION

Birth defects caused by maternal diabetes are a major health problem. It has been suggested that embryonic malformation is associated with oxidative stress, which induces excessive cell death in embryo and yolk sac. A number of factors, such as PKC, cPLA.sub.2, and MAPKs have been implicated in diabetic embryopathy. However, the roles of these factors in embryonic malformation and the mechanisms by which these factors regulate reactive oxygen species production and apoptosis are largely unknown.

Results presented herein indicate that exposure to maternal hyperglycemia induces both an increase in oxidative stress and a decrease in glutathione-mediated antioxidant defense in developing embryos. This in turn causes aberrations in stress-activated MAPK signaling pathways and membrane phospholipids signal transduction pathways, and result in excessive apoptosis and dysmorphogenesis. In addition, there is biochemical deficiencies in membrane lipids (arachidonic acid and myoinositol) and excess reactive oxygen species.

These findings suggest that hyperglycemia activates PKC in the cell. Activated PKC activates cPLA.sub.2, leading to lipid peroxidation and increased production of reactive oxygen species. Elevated oxidative stress alters MAPK activation, possibly by increasing JNK activity. Increased JNK activity promotes release of cytochrome C from mitochondria, which activates caspase 9 and caspase 3, leading to apoptosis. The roles and mechanisms of these factors and signaling pathways in diabetic embryopathy can be examined using an in vivo maternal diabetic animal model and in vitro whole embryo and cell cultures combined with contemporary cellular and molecular biological techniques. Data obtained from these experiments will provide important information for developing therapeutic approaches to prevent birth defects.

It is hypothesized that nutritional supplements, especially antioxidants, can inhibit and/or reduce the rates of apoptosis and malformation in embryos under hyperglycemic conditions. Choice of supplements is based on the ability of each supplement to correct the following hyperglycemia-associated abnormalities: increased reactive oxygen species generation, abnormal membrane phospholipid metabolism, and decreased glutathione synthesis (FIG. 9, see Original Patent).

For example, three sets of supplements are selected to correct three different levels of hyperglycemia-induced aberrations that contribute to diabetic embryopathy. The first supplement set is a combination of two potent antioxidants, lipoic acid and vitamin C, and is designed to reduce reactive oxygen species-mediated effects and to support reductive regeneration of other antioxidants. Lipoic acid and its reduced form, dihydrolipoic acid, are thiol antioxidants that are soluble in both aqueous and membrane compartments and function as scavengers of hydroxyl radicals and singlet oxygen in membranes and cytosol. Lipoic acid has been shown to decrease oxidative stress in diabetic patients with poor glycemic control, and to reduce malformations in embryos of STZ-induced diabetic rats. Vitamin C is a potent scavenger of several reactive oxygen species including hydroxyl radical, singlet oxygen, and superoxide radical, and it intervenes to inhibit membrane lipid peroxidation. Both lipoic acid and vitamin C interact with other antioxidants to provide reducing equivalents for the regeneration of active vitamin E and GSH. Therefore, it is predicted this combination of antioxidants will increase total antioxidant capacity in diabetic mothers despite their ongoing hyperglycemia.

The second supplement set is a combination of vitamin E and arachidonic acid, and is designed to prevent membrane lipid peroxidation and improve aberrant membrane lipid metabolism associated with hyperglycemia. Vitamin E is a major lipid-soluble membrane antioxidant that acts as a peroxidation chain-breaking agent to prevent loss of membrane function and integrity. Vitamin E supplementation of children with type 1 diabetes reduced erythrocyte lipid peroxidation. It has been shown that supplementing the diets of diabetic rats with arachidonic acid reduces malformation rates in vitro and in vivo. By combining vitamin E and arachidonic acid supplementation, it is anticipated that an interactive effect on membrane structure and function will reduce malformation rates to a greater extent than either alone.

The third supplement set combines glutathione precursor N-acetylcysteine (NAC), with the methionine/cysteine precursor folic acid and is designed to support and augment glutathione synthesis in diabetic mothers. Analysis of intracellular metabolites in the glutathione pathway indicated that malformed embryos and yolk sacs were severely depleted of glutathione (GSH) antioxidant capacity (2-4-fold reduction in GSH/GSSG). Results presented above indicated a decrease in methionine and an increase in homocysteine, consistent with a deficiency in functional folate induced by chronic oxidative stress and an increased requirement for glutathione. The increase in cysteine in malformed embryos and yolk sacs strongly supports the hypothesis that .gamma.-glutamylcysteine synthetase expression and activity are reduced during hyperglycemia-induced oxidative stress. It is expected that supplementation with folic acid will increase methionine, decrease homocysteine, and increase GSH synthesis in diabetic mothers.

Individual supplement sets may reduce, but not completely prevent, malformations and that a combination of two or more supplement sets may reduce malformation rates to background levels in diabetic mothers. Identification of metabolic and molecular endpoints that are significantly shifted by supplementation toward the levels in normal embryos and yolk sacs will provide strong evidence to suggest that these alterations are the most significant in the genesis of diabetic embryopathy. The comparison of the magnitude of these changes between embryos and yolk sacs is expected to provide important insights into the relative importance of each in the development and prevention of malformations.

In one embodiment, the present invention provides a dietary supplement and a method of using such supplement to reduce birth defect caused by maternal diabetes. The dietary supplement comprises (a) an effective amount of a source of lipoic acid; (b) an effective amount of a source of vitamin C; (c) an effective amount of a source of vitamin E; (d) an effective amount of a source of arachidonic acid; (e) an effective amount of a source of folic acid; and (f) an effective amount of a source of glutathione precursor N-acetylcysteine.

It is known that myoinositol when administered in combination with vitamin E and arachidonic acid reduces embryonic defect rate in diabetes related pregnancy. It is also speculated that under hyperglycemic conditions there is a widespread relative intracellular myoinositol deficiency in man, which suggests that restoration of normal intracellular myoinositol concentrations might prove to benefit in the prevention and treatment of certain complications because of diabetes. Accordingly it is contemplated that an effective amount of myoinositol may be added in the instant dietary supplement to reduce birth defect caused by maternal diabetes.

Preferably, the effective amount of lipoic acid is from about 1 mg to about 100 mg per day; more preferably, the effective amount of lipoic acid is from about 5 mg to about 20 mg per day. The effective amount of vitamin C is from about 10 mg to about 500 mg per day; more preferably, the effective amount of vitamin C is from about 75 mg to about 150 mg per day. The effective amount of vitamin E is from about 50 IU to about 500 IU per day; more preferably, the effective amount of vitamin E is from about 200 IU to about 300 IU per day. The effective amount of Safflower oil, the source of arachidonic acid that comprises at least 79% of linoleic acid, is 1-100 ml daily; more preferably 5-10 ml daily. The effective amount of N-acetylcysteine is from about 50 mg to about 750 mg three times a day; more preferably, the effective amount of N-acetylcysteine is from about 250 mg-500 mg three times a day. The effective amount of and the effective amount of [6S]-5-methyltetrahydrofolate (MTHF), one of the derivative folic acid, is from about 100 .mu.g to about 800 .mu.g per day.

In general, the dietary supplement is administered to an individual before or during pregnancy. The supplement can be administered by a suitable means of consumption generally known and practiced in the art. For example, the supplement can be taken as a tablet or capsule which can be easily swallowed, chewed, or dissolved. Alternatively, the dietary supplement can be formulated into a powder or liquid for convenient addition to drinks, baked goods, dairy products or other food stuffs. The dietary supplement can also be consumed in the form of a snack bar, drink or lozenge. The supplement preferably is consumed on a daily basis, e.g., once a day. The dietary supplement can also be consumed multiple (greater than one) times a day.

In another embodiment, there are provided a dietary supplement and a method of using such supplement to reduce birth defect caused by maternal diabetes. The dietary supplement comprises one or more antioxidants, one or more compounds that improve membrane lipid metabolism, and one or more compounds that support glutathione synthesis. In general, the antioxidants can be vitamin C, vitamin E and lipoic acid. Examples of compounds that improve membrane lipid metabolism include arachidonic acid, and compounds that support glutathione synthesis include methionine/cysteine precursor (e.g. folic acid) and glutathione precursor (e.g. N-acetylcysteine). This supplement may further contain an effective amount of myoinositol. These various nutrients can be formulated and administrated as that described above.
 

Claim 1 of 14 Claims

1. A dietary supplement for reducing birth defects caused by maternal diabetes consisting of: (a) an effective amount of lipoic acid; (b) an effective amount of vitamin C; (c) an effective mount of vitamin E; (d) an effective amount of arachidonic acid; (e) an effective amount of a folic acid; and (f) an effective amount of N-acetylcysteine.

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