Application of cell-free fetal DNA for early evaluation of preeclampsia to reduce maternal mortality by low-cost method – A prospective cohort study

Adverse pregnancy outcomes such as preeclampsia are the leading cause of maternal morbidity and mortality in the world and its incidence is increasing. It has been observed in some studies that cell-free fetal DNA (cff DNA) is increased in maternal serum associated with preeclampsia. In the present study, we have tested whether the elevated amount of cff DNA in maternal plasma is associated with PE and development of new marker by the low-cost method to predict preeclampsia. Twenty-one pregnant women within the age group of 20–30 years attending for routine antenatal checkups at (G & O) antenatal OPD after 20 weeks with fulfilling the diagnostic criteria of preeclampsia were included in our study. Age-matched pregnant women without hypertension were included as controls. A complete clinical history and anthropometric observation showed that gravida (total number of pregnancy in a patient including present pregnancy), gestational age, gestational age at birth, birth weight in preeclampsia subjects were non-significantly lower than normotensive subjects. Blood analysis showed lower platelet count and higher creatinine level, bilirubin level, and liver enzyme activities in preeclampsia subjects in comparison to normotensive subjects. Identification of cell-free fetal DNA (cff DNA) in maternal plasma by using two in-house methods (phenol-chloroform-isopropanol and NaI) was found comparable and its content (GE/µL) in preeclampsia subjects were significantly higher than the normotensive subjects. Correlation analysis showed that APGAR score was significantly negatively correlated with both systolic and diastolic blood pressure and significantly positively correlated with gestational age and gestational age at birth; whereas, cff DNA was significantly positively correlated with blood pressure but significantly negatively correlated with platelet count. In conclusion, our study demonstrated that APGAR score, which is one of the indicators of physiologic maturity of the infant is severely affected by the causative factors of preeclampsia and cell-free fetal DNA quantification may be a promising marker for future adverse pregnancy outcome.

Trianthema portulacastrum L. extract protects against gamma radiation induced human red blood cell membrane damage in vitro

Radiation-induced oxidation of membrane lipids and proteins appear to be responsible for damaging the red cell membranes. The membrane integrity of circulating red blood cells (RBCs) is compromised by the deleterious action of γ-radiation in humans. Trianthema portulacastrum L. is an inexpensive, non-toxic herb commonly used in ayurvedic medicine due to its significant pharmacological activities. Methanolic extract of different parts of T. portulacastrum L. contain carbohydrates, polyphenols, flavonoids, alkaloids and terpenoids; whereas saponin is present only in root extract, while cardiac glycosides are absent in root extract. Here, we explored the radio-protective role of leaf, stem, root and whole plant extracts of T. portulacastrum L. against the γ-radiation induced membrane damage of human red blood cells (RBCs). The RBCs on γ-irradiation (4 Gy) exposure showed elevated TBARS level while inhibited ATPase activities in the membrane ghosts. Treatment of RBCs-with different extracts of T. portulacastrum L. at different doses (50 µg/mL, 100 µg/mL, 200 µg/mL) 1h prior to the exposure of γ-radiation significantly mitigated these changes in the RBC membranes due to presence of antioxidants in the extracts.

Grape extract protects against ionizing radiation-induced membrane alterations in Red Blood Cells.

Ionizing radiation causes damage to biomolecules in living cells through oxidative stress by excessive generation of reactive oxygen species (ROS) from radiolysis of body water. Blood and its components including the cells are exposed to a significant dose of radiation during irradiation. Grapes (Vitis vinifera L.) contain several bioactive phytochemicals and are rich source of antioxidants. Therefore, we hypothesized that the grape extracts would offer protection against the ionizing radiation-induced damage of the red blood cells (RBCs). To test our hypothesis, in the current study we investigated the radio-protective actions of extract of four different grape (Vitis vinifera) cultivars, namely Flame seedless (Black grapes), Kishmish chorni (Black with reddish brown), Red globe (Red) and Thompson seedless mutant (Sonaka, Green) against the g-irradiation-induced oxidative stress leading to the structural alteration in the RBC membrane in vitro. Freshly drawn blood samples from healthy volunteers itself or mixed with grape extracts from seed, skin or pulp of each cultivar were irradiated at 4 Gy after one hour of treatment. -irradiation for one hour did not change the hematological parameters. The average osmotic fragility (H50) and the maximum rate of hemolysis (dH/dC)max increased after the -irradiation. The confocal microscopic and atomic force microscopic (AFM) studies showed that irradiation induced transformation of RBC from biconcave cells to echinocytes, altered their surface roughness and the vertical distance. The grape extracts did not alter the viability of human erythrocytes. Our results suggested that the grape extract pretreatment ameliorated the ionizing radiation-induced alterations at a dose of 4 Gy in human erythrocytes in vitro. Moreover, protection offered by the seed extract was significantly better than that that of skin or pulp of the same cultivar. Furthermore, the protective action of grape extract depends on its source (seed, skin or pulp) as well on cultivars.

Black tea extract protects against -radiation-induced membrane damage of human erythrocytes.

The membrane integrity of circulating red blood cells (RBCs) is compromised by the deleterious actions of -radiation in humans. Tea is the most widely consumed popular, inexpensive and non-toxic beverage rich in antioxidants. Here, we explored the radioprotective actions of black tea against the -radiation-induced membrane permeability of human erythrocytes. The phytochemical analysis of tea revealed the total polyphenol content to be 114.89±6.03 mg gallic acid equivalent/g dry wt. and flavonoid content, 34±0.11 mg catechin equivalent/g dry wt. of the extractable solid in the commercially available tea bags. Tea extracts showed potential scavenging of H2O2 and NO, appreciable extent of total antioxidant capacity and effective anti-hemolytic action. Tea extracts (15 µg/mL) significantly ameliorated the -radiation-induced increase of the levels of thiobarbituric acid-reactive substances (TBARS, an index of lipid peroxidation) in the RBC membrane ghosts. Stored blood showed higher levels of K+ ion as compared to the normal blood which was elevated by -radiation. Membrane ATPase was inhibited by the exposure to -radiation. Treatment of RBCs with the tea extracts (15 µg/ml) prior to the exposure of -radiation significantly mitigated these changes in the erythrocyte membranes caused by the lower dose of radiation (4 Gy) as compared to that induced by the higher dose of -radiation.

Scavenging and antioxidant properties of different grape cultivars against ionizing radiation-induced liver damage ex vivo.

Ionizing radiation (IR) has become an integral part of the modern medicine— both for diagnosis as well as therapy. However, normal tissues or even distant cells also suffer IR-induced free radical insult. It may be more damaging in longer term than direct radiation exposure. Antioxidants provide protection against IR-induced damage. Grapes are the richest source of antioxidants. Here, we assessed the scavenging properties of four grape (Vitis vinifera) cultivars, namely Flame seedless (Black), Kishmish chorni (Black with reddish brown), Red globe (Red) and Thompson seedless mutant (Green), and also evaluated their protective action against -radiation-induced oxidative stress in liver tissue ex vivo. The scavenging abilities of grape seeds [2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50=0.008±0.001 mg/mL), hydrogen peroxide (IC50=0.49 to 0.8 mg/mL), hydroxyl radicals (IC50=0.08±0.008 mg/mL), and nitric oxide (IC50=0.8±0.08 mg/mL)] were higher than that of skin or pulp. Gamma () radiation exposure to sliced liver tissues ex vivo from goat, @ 6 Gy significantly (P <0.001) decreased reduced glutathione (GSH) content by 21.2% and also activities of catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione s-transferase (GST) by 49.5, 66.0, 70.3, 73.6%, respectively. However, it increased thiobarbituric acid reactive substances (TBARS) by 2.04-fold and nitric oxide level by 48.6% compared to untreated group. Further increase in doses (10 or 16 Gy) of -radiation correspondingly decreased GSH content and enzyme activities, and increased TBARS and nitric oxide levels. Grape extract treatment prior to ionizing radiation exposure ameliorated theses effects at varying extent. The seed extracts exhibited strong antioxidant potential compared to skin or pulp extracts of different grape cultivars against oxidative damage by ionizing radiation (6 Gy, 10 Gy and 16 Gy) in sliced liver tissues ex vivo. Grape extracts at higher concentration (10 mg extract/g liver tissue) showed stronger antioxidant potential against lower dose (6 Gy) of ionizing radiation. Our results suggest that grape extracts could serve as a potential source of natural antioxidant against lower doses of IR-induced oxidative stress in liver extracts ex vivo.

Grapevine fruit extract protects against radiation-induced oxidative stress and apoptosis in human lymphocyte.

Ionizing radiation (IR) causes oxidative stress through overwhelming generation of reactive oxygen species (ROS) in the living cells leading the oxidative damage further to biomolecules. Grapevine (Vitis vinifera L.) posses several bioactive phytochemicals and is the richest source of antioxidants. In this study, we investigated V. vinifera for its phytochemical content, enzymes profile and, ROS- and oxidant-scavenging activities. We have also studied the fruit extract of four different grapevine viz., Thompson seedless, Flame seedless, Kishmish chorni and Red globe for their radioprotective actions in human lymphocytes. The activities of ascorbic acid oxidase and catalase significantly (P <0.01) differed among extracts within the same cultivar, while that of peroxidase and polyphenol oxidase did not differ significantly. The superoxide radical-scavenging activity was higher in the seed as compared to the skin or pulp of the same cultivar. Pretreatment with grape extracts attenuated the oxidative stress induced by 4 Gy γ-radiation in human lymphocytes in vitro. Further, γ-radiation-induced increase in caspase 3/7 activity was significantly attenuated by grape extracts. These results suggest that grape extract serve as a potential source of natural antioxidants against the IR-induced oxidative stress and also inhibit apoptosis. Furthermore, the protective action of grape depends on the source of extract (seed, skin or pulp) and type of the cultivars.

Challenges of Ionizing Radiation in Tumor Treatment and Role of Angiogenesis.

Ionizing radiation is a non-specific, but most widely used therapeutic method for cancer treatment. However, a minor fraction of tumor cell population manages to survive after radiation. Radiation efficacy depends on adequate oxygen supply. Rapid growing tumors cause hypoxia that upregulates many pro-survival pathways. At clinical doses, radiation activates inflammatory pathways and causes oxidative stress that plays a positive role during angiogenesis. Selective targeting of signaling mechanisms may radiosensitize tumors.

Antioxidant potential of different grape cultivars against Fenton-like reagent-induced liver damage ex-vivo.

The phytochemicals present in the grapes are responsible for nutraceutical and health benfits due to their antioxidant properties. These phytochemicals, however, vary greatly among different cultivars. In this study, we evaluated the antioxidant potential and protective role of four different Indian grape (Vitis vinifera) cultivars extracts, namely Flame seedless (Black grapes), Kishmish chorni (Black with reddish brown), Red globe (Red) and Thompson seedless mutant (Sonaka, Green) against the Fenton-like reagent (200 mmole H2O2, 2 mmole ascorbate, 25 mmole FeSO4)-induced liver damage. Non-enzymatic antioxidants, such as glutathione (GSH) levels and activities of antioxidant enzymes, such as glutathione S-transferase (GST) and superoxide dismutase (SOD), as well as total antioxidant capacity (TAC) were highest in the grape seed, followed by skin and pulp. Among edible parts of different cultivars, skin of Flame seedless (Black) cultivar showed highest antioxidant potential, while the Thompson seedless the least potential. These antioxidants were found to be significantly (P<0.01) correlated with the levels of total phenol, flavonoids and ascorbic acid. Fenton-like reagent treatment significantly (P<0.001) decreased GSH content by 39.1% and activities of catalase (CAT) by 43.2% and glutathione reductase (GR) by 60%, while increasing thiobarbituric acid reactive substances (TBARS) and nitric oxide levels by 2.13-fold and 0.64-fold, respectively and GST activity by 0.81-fold. Pre-treatment with grape seed extracts showed the best hepatoprotective action against Fenton-like reagent-induced damage, followed by the extracts of skin and pulp of any cultivar. Thus, our study showed the significant amounts of antioxidants were in grape seed, followed by its skin and pulp, which varied among the cultivars and was associated with the protective action of grape extracts against Fenton-like reagent-induced liver damage ex-vivo.

Role of nitric oxide, angiogenic growth factors and biochemical analysis in preeclampsia.

Preeclampsia, a pregnancy-related hypertensive disorder, is one of the leading causes of fetal and maternal mortality and morbidity globally. Angiogenic growth factors, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are involved in the generation of new blood vessels required for placental development and physiological functions, while nitric oxide (NO) acts as vasodilator and also plays a role in angiogenesis. The objective of this study was to evaluate the role of NO, angiogenic growth factors (VEGF and PIGF) and other biochemical parameters in the development of preeclampsia among pregnant mothers. A complete clinical history, including anthropometric measurements and biochemical investigations, including renal function tests, liver function tests and lipid profile were performed among twenty preeclampsia patients aged 19 to 32 yrs. Results were compared with age-matched normotensive pregnant mothers. The body weight, body mass index (BMI), blood pressure, concentrations of urea, uric acid and triglyceride and activities of transaminase enzymes (aspartate transaminase, AST and alanine transaminase, ALT) in serum were significantly higher (p<0.05) than normotensive subjects. Serum concentrations of VEGF, PlGF and NO were significantly decreased (p<0.005) in preeclamptic patients. NO was found negatively correlated with body weight (r = -0.369, p<0.05), systolic blood pressure (r = -0.822, p<0.005), diastolic blood pressure (r = -0.714, p<0.005) and was positively correlated with VEGF (r = 0.464, p<0.005) and PlGF (r = 0.546, p<0.005). VEGF and PlGF showed significant (p<0.005) negative correlation with systolic and diastolic blood pressure and PlGF was significantly correlated with triglyceride (r = -0.379). However, no significant correlation was observed between the VEGF and PlGF. In conclusion, the results indicated that body weight, triglyceride, angiogenic growth factors and NO might associate with preeclampsia development.

Dietary grapes (Vitis vinifera) feeding attenuates ethanol-induced oxidative stress in blood and modulates immune functions in mice.

Ethanol metabolism is known to induce overwhelming production of reactive oxygen species (ROS) and also to cause associated immune dysfunction. Several interventional agents of plant origin, in particular fruits and vegetables have been used to counteract these alterations induced by ethanol. In this study, we investigated the efficacy of dietary feeding of skin and flesh of grapes (Vitis vinifera L.) on the alterations in immune and vascular functions in mice with liver abnormalities induced by chronic ethanol consumption. Results revealed that feeding of both grape skin and flesh (2.5 g/kg body wt/day) effectively attenuated the oxidative stress and alterations in immune function and angiogenesis induced by chronic ethanol consumption (1.6 g/kg body wt/day for 12 weeks) in mice. The antioxidant actions of the grape skin and flesh as observed in this study might be attributed to the polyphenols present in the grapes.

Effects of long-term ethanol consumption on adhesion molecules in liver.

Adhesion molecules play an important role in the pathogenesis of several diseases. In this study, expression of adhesion molecules was examined in the setting of chronic alcohol induced liver damage of male albino Wistar strain rats (16-18 weeks-old, 200-220 g) in a time dependent manner. Decreased protein level and increased activities of liver marker enzymes in response to the chronic ethanol (1.6 g ethanol/kg body weight/day) exposure, indicated that these animals suffered from liver damage in a time-dependent manner. Flow cytometric analysis revealed that chronic ethanol treatment induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression in liver tissues of rats with duration of ethanol exposure. The results suggest that the adhesion molecules may be associated with the initiation of hepatic injury during alcohol intoxication.

Protective effect of resveratrol and vitamin E against ethanol-induced oxidative damage in mice: Biochemical and immunological basis.

The metabolism of ethanol gives rise to the generation of excess amounts of reactive oxygen species and is also associated with immune dysfunction. We examined the efficacy of resveratrol and vitamin E on the immunomodulatory activity and vascular function in mice with liver abnormalities induced by chronic ethanol consumption by measuring the protein, liver-specific transaminase enzymes, antioxidant enzymes and non-enzymes such as reduced glutathione (GSH) content, thiobarbituric acid reactive substance (TBARS) level, nitrite level, and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and cytokines such as interleukin (IL)-2, IL-4, IL-10, tumor necrosis factor (TNF)-, gamma interferon (IFN-), vascular endothelial growth factor (VEGF)-A and transforming growth factor (TGF)-1 in mice blood. Ethanol (1.6 g/kg body wt/day) exposure for 12 wks significantly increased TBARS and nitrite levels and GST activity, and significantly decreased GSH content and the activities of SOD, CAT, GR and GPx in whole blood hemolyzate of 8-10 wks-old male BALB/c mice (weighing 20-30 g). Ethanol exposure also elevated the activities of transaminase enzymes (AST and ALT), IL-10, TNF-, IFN-, VEGF-A and TGF-1, while decreasing the albumin concentration and IL-4 activity in the serum. Both resveratrol (5 mg kg-1 day-1) and vitamin E (80 mg kg-1 day-1) treatment significantly reduced AST, ALT, GST, IL-10, TNF-, IFN-, VEGF-A and TGF-1 activities and levels of TBARS and nitrite, and elevated albumin content, GSH level and activities of SOD, CAT, GR and GPx, compared to ethanol-treated group. Thus, results from the study demonstrated that both resveratrol (5 mg kg-1 day-1) and vitamin E (80 mg kg-1 day-1) can effectively ameliorate ethanol (1.6 g kg-1 day-1)-induced oxidative challenges, immunomodulatory activity and angiogenesis processes.

Time-dependent effects of ethanol on blood oxidative stress parameters and cytokines.

Alcohol consumption is implicated in the genesis of a spectrum of liver abnormalities, which are associated with a number of factors. In the present study, time-dependent effects of ethanol on cytokines (TNF-alpha, IL-2, IL-4, IL-10, IFN-gamma, VEGF-A and TGF-beta1) in serum, and blood oxidative stress parameters such as reduced glutathione content, TBARS level and activities of GPx, GR, GST, catalase and SOD in 8-10 weeks-old male BALB/c mice have been investigated. Ethanol administered @ 1.6 g/kg body wt/day significantly increased the activities of liver marker enzymes AST, ALT and ALP. Serum nitrite levels and haemolysate TBARS level also increased, while total antioxidant status in serum and GSH content in whole blood hemolysate decreased from 4th week onwards of exposure. In spite of the increased serum nitrite level and GST activity in the haemolysate, albumin level in serum, GPx and GR activities in haemolysate decreased after 12 weeks of exposure. Chronic ethanol treatment did not show any effect on IL-2, but IL-4 level was reduced and other cytokines such as IL-10, TNF-alpha, IFN-gamma, TGF-beta1 and VEGF-A levels were increased significantly after 12 weeks. The study indicates a relationship between free radical generation and immune response, and suggests that ethanol-induced liver damage is associated with oxidative stress and immunological alterations in a time-dependent manner.

Effect of lecithin with vitamin-B complex and tocopheryl acetate on long-term effect of ethanol induced immunomodulatory activities.

The alcoholic liver disease usually causes overall immunological alterations which might be attributed to hepatic disease, to ethanol action, and/or to malnourishment. In the present study, efficacy of lecithin with vitamin-B complex to treat ethanol induced immunomodulatory activity was compared with the effect of lecithin alone and tocopheryl acetate (vitamin E). Ethanol (1.6 g/kg body wt/day for 12 weeks) exposure increased thiobarbituric acid reactive substance (TBARS) level, while decreased superoxide dismutase (SOD) activity and reduced glutathione (GSH) content in whole blood hemolysate of 8-10 week-old male BALB/c mice (weighing 20-30 g). The activities of transaminase (AST and ALT) enzymes, interleukin (IL)-10 and gamma interferon (IFN-gamma) elevated, while IL-2 and IL-4 reduced in mice serum due to ethanol exposure. These suggested that oxidative stress and immunomodulatory activities were interdependent and associated with ethanol induced liver damage. Lecithin treatment significantly reduced AST (32.44%), ALT (32.09%), IL-10 (25.63%) activities and TBARS content (12.76%) compared to ethanol treated group. However, lecithin with vitamin-B complex treatment, significantly reduced AST (62.83%); ALT (61.96%); IL-10 (35.88%); IFN-gamma (22.55%) activities and TBARS content (31.58%), while significantly elevated GSH content (36.49%) and SOD activity (61.21%). Tocopheryl acetate treatment significantly reduced AST (62.83%); ALT (61.54%); IL-10 (36.35%): IFN-gamma (23.28%) activities and TBARS content (35.84%). while significantly elevated GSH content (28.76%) and SOD activity (62.42%) compared to ethanol treated group. These findings persuasively argued that lecithin with vitamin-B complex was a new promising therapeutic approach in controlling ethanol induced immunomodulatory activities involving liver damage processes. Prevention of oxidative stress with correction of nutritional deficiency caused alteration in the ethanol-induced immunomodulatory activities and associated liver diseases.

Modulation of lecithin activity by vitamin-B complex to treat long term consumption of ethanol induced oxidative stress in liver.

Alcoholic liver disease (ALD) develops as a consequence of priming and sensitizing mechanisms rendered by cross-interactions of primary mechanistic factors and secondary risk factors. Chronic alcohol abuse and its progression to ALD are associated with abnormal metabolism and low tissue or plasma levels, or both, of many micronutrients. Glutathione depletion is considered the most important sensitizing mechanism. In the present study efficacy of lecithin with vitamin-B complex to treat ethanol induced oxidative stress was compared with the effect of lecithin alone, tocopheryl acetate (vitamin E), as well as capacity of hepatic regeneration during abstention. Ethanol (1.6g / kg body weight/ day for 4 weeks) affects body weight in 16-18 week old male albino rats of Wistar strain weighing 200-220 g. Thiobarbituric acid reactive substance level, nitrite content, protein carbonyl group level, redox ratio (oxidized to reduced glutathione ratio), superoxide dismutase activity, and glutathione s-transferase activity significantly increased on ethanol exposure. Whereas reduced glutathione content, and activities of catalase, glutathione reductase and glutathione peroxidase significantly reduced due to ethanol exposure. These changes were reversed by different treatment. The results suggest that tocopheryl acetate (vitamin E) could partially reverse these changes and act as a potential therapeutic agent. However, lecithin with vitamin-B complex treatment is a promising therapeutic approach. Furthermore, preventive measures were more effective than curative treatment. Prevention of oxidative and nitrosative stress along with correction of nutritional deficiency is one of the proposed mechanisms for the therapeutic approach.

Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver.

The production of reactive oxygen species (ROS) is considered to be a major factor in oxidative cell injury. The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against such hepatic damage. Silymarin, derived from the milk thistle plant, Silybium marianum, has been used in traditional medicine as a remedy for diseases of the liver and biliary tract. In the present study, the effect of hepatoprotective drug silymarin on body weight and biochemical parameters, particularly, antioxidant status of ethanol-exposed rats was studied and its efficacy was compared with the potent antioxidant, ascorbic acid as well as capacity of hepatic regeneration during abstention. Ethanol, at a dose of 1.6 g/kg body wt/day for 4 wks affected body weight in 16-18 week-old male albino rats (Wistar strain weighing 200-220 g). Thiobarbituric acid reactive substance (TBARS) level, superoxide dismutase (SOD), and glutathione-s-transferase (GST) activities were significantly increased, whereas GSH content, and catalase, glutathione reductase (GR) and GPx (glutathione peroxidase) activities significantly reduced, on ethanol exposure. These changes were reversed by silybin and ascorbic acid treatment. It was also observed that abstinence from ethanol might help in hepatic regeneration. Silybin showed a significant hepatoprotective activity, but activity was less than that of ascorbic acid. Furthermore, preventive measures were more effective than curative treatment.

Alcohol: its health and social impact in India.

Alcoholic beverages have been used in human societies since the beginning of recorded history. The patterns of alcohol intake around the world are constantly evolving, and alcohol is ubiquitous today. Research has contributed substantially to our understanding of the relation of drinking to specific disorders, and has shown that the relation between alcohol consumption and health outcomes is complex and multidimensional. Increases in the average volume of drinking are predicted for the most populous regions of the world in Southeast Asia including India. Cultural differences apparently influence the pattern of alcohol consumption. In addition, alcohol is linked to categories of disease whose relative impact on the global burden is predicted to increase. Therefore, it is appropriate to implement policies with targeted harm reduction strategies. The crucial need, from a public health perspective, is for regular means of coordination whereby prevention of alcohol-related problems is taken fully into account in policy decisions about alcohol control and regulation in the market for alcoholic beverages.