Epigenetic effects of folate and related B vitamins on brain health throughout life: Scientific substantiation and translation of the evidence for health improvement strategies.

Suboptimal status of folate and/or interrelated B vitamins (B12 , B6 and riboflavin) can perturb one-carbon metabolism and adversely affect brain development in early life and brain function in later life. Human studies show that maternal folate status during pregnancy is associated with cognitive development in the child, whilst optimal B vitamin status may help to prevent cognitive dysfunction in later life. The biological mechanisms explaining these relationships are not clear but may involve folate-related DNA methylation of epigenetically controlled genes related to brain development and function. A better understanding of the mechanisms linking these B vitamins and the epigenome with brain health at critical stages of the lifecycle is necessary to support evidence-based health improvement strategies. The EpiBrain project, a transnational collaboration involving partners in the United Kingdom, Canada and Spain, is investigating the nutrition-epigenome-brain relationship, particularly focussing on folate-related epigenetic effects in relation to brain health outcomes. We are conducting new epigenetics analysis on bio-banked samples from existing well-characterised cohorts and randomised trials conducted in pregnancy and later life. Dietary, nutrient biomarker and epigenetic data will be linked with brain outcomes in children and older adults. In addition, we will investigate the nutrition-epigenome-brain relationship in B vitamin intervention trial participants using magnetoencephalography, a state-of-the-art neuroimaging modality to assess neuronal functioning. The project outcomes will provide an improved understanding of the role of folate and related B vitamins in brain health, and the epigenetic mechanisms involved. The results are expected to provide scientific substantiation to support nutritional strategies for better brain health across the lifecycle.

Emerging roles for folate and related B-vitamins in brain health across the lifecycle.

Nutrition plays a fundamental role in supporting the structural and functional development of the human brain from conception, throughout early infancy and extending into later life. A growing body of evidence suggests that folate and the metabolically related B-vitamins are essential for brain health across all age groups, owing to their specific roles in C1 metabolism and particularly in the production of S-adenosylmethionine, a universal methyl donor essential for the production of neurotransmitters. Emerging, though not entirely consistent, evidence suggests that maternal folate status throughout pregnancy may influence neurodevelopment and behaviour of the offspring. Furthermore optimal B-vitamin status is associated with better cognitive health in ageing. Of note, a recent clinical trial provided evidence that supplementation with folic acid and related B-vitamins over a 2-year-period reduced global and regional brain atrophy, as measured by MRI scan in older adults. In terms of potential mechanisms, the effects of these B-vitamins on cognitive health may be independent or may be mediated by nutrient-nutrient and/or relevant gene-nutrient interactions. Furthermore, a new area of research suggests that the in utero environment influences health in later life. Folate, an important cofactor in C1 metabolism, is indirectly involved in DNA methylation, which in turn is considered to be one of the epigenetic mechanisms that may underlie fetal programming and brain development. The present review will explore the evidence that supports a role for folate and the related B-vitamins in brain health across the lifecycle, and potential mechanisms to explain such effects.

MTHFR 677TT genotype and disease risk: is there a modulating role for B-vitamins?

Methylenetetrahydrofolate reductase (MTHFR) is a critical folate-metabolising enzyme which requires riboflavin as its co-factor. A common polymorphism (677C→T) in the MTHFR gene results in reduced MTHFR activity in vivo which in turn leads to impaired folate metabolism and elevated homocysteine concentrations. Homozygosity for this polymorphism (TT genotype) is associated with an increased risk of a number of conditions including heart disease and stroke, but there is considerable variability in the extent of excess risk in various reports. The present review will explore the evidence which supports a role for this polymorphism as a risk factor for a number of adverse health outcomes, and the potential modulating roles for B-vitamins in alleviating disease risk. The evidence is convincing in the case which links this polymorphism with hypertension and hypertensive disorders of pregnancy, particularly preeclampsia. Furthermore, elevated blood pressure was found to be highly responsive to riboflavin intervention specifically in individuals with the MTHFR 677TT genotype. Future intervention studies targeted at these genetically predisposed individuals are required to further investigate this novel gene-nutrient interaction. This polymorphism has also been associated with an increased risk of neural tube defects (NTD) and other adverse pregnancy outcomes; however, the evidence in this area has been inconsistent. Preliminary evidence has suggested that there may be a much greater need for women with the MTHFR 677TT genotype to adhere to the specific recommendation of commencing folic acid prior to conception for the prevention of NTD, but this requires further investigation.

B-vitamins, homocysteine metabolism and CVD.

The present review focuses on the B-vitamins, i.e. folate, vitamin B12, vitamin B6 and riboflavin, that are involved in homocysteine metabolism. Homocysteine is a S-containing amino acid and its plasma concentrations can be raised by various constitutive, genetic and lifestyle factors, by inadequate nutrient status and as a result of systemic disease and various drugs. Hyperhomocysteinaemia is a modest independent predictor of CVD and stroke, but causality and the precise pathophysiological mechanism(s) of homocysteine action remain unproven. The predominant nutritional cause of raised plasma homocysteine in most healthy populations is folate insufficiency. Vitamin B12 and, to a lesser extent, vitamin B6 are also effective at lowering plasma homocysteine, especially after homocysteine lowering by folic acid in those individuals presenting with raised plasma homocysteine. However, riboflavin supplementation appears to be effective at lowering plasma homocysteine only in those individuals homozygous for the T allele of the C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene. This gene codes for the MTHFR enzyme that produces methyltetrahydrofolate, which, in turn, is a substrate for the remethylation of homocysteine by the vitamin B12-dependent enzyme methionine synthase. Individuals with the MTHFR 677TT genotype are genetically predisposed to elevated plasma homocysteine, and in most populations have a markedly higher risk of CVD.

Acute absorption of folic acid from a fortified low-fat spread.

OBJECTIVE: To explore the feasibility of low-fat spreads as vehicles for folic acid (FA) fortification by determining the acute absorption of FA from a fortified spread. DESIGN: Double blind, crossover study to test each of the following treatments administered at 1-weekly intervals: (A) 20 g low-fat (40%) spread fortified with 200 microg FA and a placebo tablet; (B) 20 g low-fat placebo spread and a 200 microg FA tablet; (C) 20 g low-fat placebo spread and a placebo tablet. SUBJECTS: A total of 13 male volunteers, aged 31.8+/-13.2 y. MAIN OUTCOME MEASURES: Plasma total folate concentrations, measured before and up to 10 h after each treatment (n=10 samples per treatment). RESULTS: Plasma folate concentrations were significantly increased compared with baseline values 1 h after administration of the FA tablet, and 1.5 h after the FA spread, and remained significantly higher than the baseline values for up to 7 h after both treatments. The maximum plasma folate response (R(max)), corrected for baseline values and 'placebo response', was established between 1 and 3 h postprandially in response to both FA spread and FA tablet, and no significant difference in R(max) was found between the two treatments (13.4 vs 14.4 nmol/l, P=0.9). The acute absorption of FA from fortified spread relative to that from the tablet, calculated on the basis of area under the plasma folate response curve, was 67% (P=0.04). CONCLUSION: The absorption of FA from fortified low-fat spread, although lower than from a tablet, is effective. These results suggest that low-fat spreads, typically associated with fat-soluble vitamin fortification, may also be considered feasible as vehicles for FA fortification.

Geographical variations in nutrition-related vascular risk factors in the UK: National Diet and Nutrition Survey of People Aged 65 Years and Over.

BACKGROUND: There is a strong north-south gradient of vascular disease in Britain, whose aetiology is not fully understood. OBJECTIVE: To test the hypothesis, in a cross-sectional survey of older people, that intakes and status indices for protective micronutrients, particularly those for which fruit and vegetables are rich sources, also vary on a north-south axis. DESIGN: The 1994-5 National Diet and Nutrition Survey of People Aged 65 Years and Over has provided a uniquely appropriate data-set for this purpose. The analysis, confined to free-living participants, compared nutrient intakes and status between people living in the north of Britain, from Scotland to Humberside, with those living south of the Wash, excluding the Midlands and Wales. RESULTS: Highly significant north-south differences, especially for vitamin C, but also to a significant extent for B-vitamins and carotenoids, indicated a more vitamin-rich diet, with more frequent use of vitamin supplements, in the south. Vitamin D status and fibre intakes were also higher in the south; sodium intake was greater in the north. Blood lipid indices did not, however, differ between north and south. North-south differences in the likelihood of receiving income support, of having manual socio-economic status and of smoking habit, appeared to be significant underlying socio-demographic factors. CONCLUSION: These findings are consistent with the hypothesis that for older British people, differences in nutrient intake and status indices between the north and south of Britain run parallel with, and may contribute to, the north-south axis of vascular disease risk.

Low-dose vitamin B-6 effectively lowers fasting plasma homocysteine in healthy elderly persons who are folate and riboflavin replete.

BACKGROUND: Current data suggest that physiologic doses of vitamin B-6 have no significant homocysteine-lowering effect. It is possible that an effect of vitamin B-6 was missed in previous trials because of a much greater effect of folic acid, vitamin B-12, or both. OBJECTIVE: The aim of this study was to investigate the effect of low-dose vitamin B-6 supplementation on fasting total homocysteine (tHcy) concentrations in healthy elderly persons who were made replete with folate and riboflavin. DESIGN: Twenty-two healthy elderly persons aged 63-80 y were supplemented with a low dose of vitamin B-6 (1.6 mg/d) for 12 wk in a randomized, double-blind, placebo-controlled trial after repletion with folic acid (400 microg/d for 6 wk) and riboflavin (1.6 mg/d for 18 wk); none of the subjects had a vitamin B-12 deficiency. RESULTS: Folic acid supplementation lowered fasting tHcy by 19.6% (P < 0.001). After folic acid supplementation, baseline tHcy concentrations ranged from 6.22 to 23.52 micromol/L and 10 subjects had suboptimal vitamin B-6 status (plasma pyridoxal-P < 20 nmol/L). Two-way analysis of variance showed that the significant improvement in vitamin B-6 status in response to vitamin B-6 supplementation (on the basis of both pyridoxal-P: and the erythrocyte aspartate aminotransferase activation coefficient) was reflected in a significant reduction in plasma tHcy of 7.5%. CONCLUSIONS: Low-dose vitamin B-6 effectively lowers fasting plasma tHcy in healthy subjects who are both folate and riboflavin replete. This suggests that any program aimed at the treatment or prevention of hyperhomocysteinemia should include vitamin B-6 supplementation.

Fluctuations in dietary methionine intake do not alter plasma homocysteine concentration in healthy men.

A moderate elevation in plasma total homocysteine (tHcy) has been established as an independent risk factor for vascular disease. An important exogenous source of homocysteine is methionine found in foods rich in animal protein. We investigated the response of tHcy to fluctuations in methionine intake in a cross-over intervention trial (two arms). Healthy men (n = 52; 19-29 y) were screened for habitual methionine intake using a food-frequency questionnaire. Subjects in the top quartile for methionine intake (n = 13), with a baseline fasting tHcy of 7.01 +/- 1.84 micromol/L (mean +/- SD), were randomly assigned to receive either a low-methionine intervention diet for 1 wk followed by a control diet for 1 wk or vice-versa. Simultaneously, those in the bottom quartile for methionine intake (n = 11), with a fasting plasma tHcy of 9.79 +/- 7. 20 micromol/L (mean +/- SD), received either a high methionine intervention diet for 1 wk followed by a control diet or vice-versa. All subjects had serum folate, red-cell folate, serum vitamin B-12 and plasma pyridoxal phosphate (PLP) concentrations within normal ranges. During the intervention, subjects in the top quartile for methionine intake reduced their daily methionine intake 79%, from 1969 +/- 639 to 407 +/- 83 mg/d (P:

Plasma pyridoxal phosphate and pyridoxic acid and their relationship to plasma homocysteine in a representative sample of British men and women aged 65 years and over.

Concentrations of pyridoxal phosphate and pyridoxic acid were measured in fasting plasma samples from British men and women aged 65 years and over, participating in a National Diet and Nutrition Survey during 1994-5, selected to be representative of the population of mainland Britain. In this population, the concentration of pyridoxal phosphate declined, whereas pyridoxic acid rose, with increasing age and frailty; however, both status indicators were strongly and directly (with a positive coefficient) correlated with estimates of vitamin B6 intake. This was little affected by the inclusion of food energy and protein intakes in the model. Forty-eight percent of the participants living in the community and 75% of those living in institutions had plasma pyridoxal phosphate concentrations below a range considered normal from other studies. In a univariate regression model, plasma pyridoxal phosphate concentrations were inversely correlated with plasma homocysteine concentrations, consistent with the hypothesis that vitamin B6 status may influence plasma homocysteine levels, and hence vascular disease risk. However, this relationship was partly attenuated in a multiple regression model including age, sex, domicile and biochemical status indices, including those of folate and vitamin B12. There was evidence that plasma pyridoxal phosphate was sensitive to metabolic conditions associated with inflammation and the acute-phase reaction, and that plasma pyridoxic acid was sensitive to renal function. Thus, neither index is an ideal predictor of vitamin B6 status in older people, unless these confounding factors are allowed for. Since poor vitamin B6 status may have health implications, e.g. for immune function, cognition, and for essential intermediary metabolic pathways in older people, it needs to be investigated as a possible public health problem.

Plasma total homocysteine response to oral doses of folic acid and pyridoxine hydrochloride (vitamin B6) in healthy individuals. Oral doses of vitamin B6 reduce concentrations of serum folate.

Plasma total homocysteine response was compared in four groups of healthy individuals given orally divided doses of vitamin supplementations for a duration of 5 weeks. The vitamin supplements; A, 0.3 mg folic acid; B, 120 mg vitamin B6; C, combination of 0.3 mg folic acid and 120 mg vitamin B6 or D, 0.6 mg folic acid reduced the concentrations of plasma total homocysteine 20, 17, 32 and 24%, respectively. However, the intergroup comparisons did not show a significant difference in the effects of vitamin supplements. Multivariate analysis with correction for differences in pre-supplement values indicated a significant effect of vitamin B6 supplementation on plasma total homocysteine and serum folate. Our data show that plasma total homocysteine concentrations are reduced with low to medium divided doses of folic acid alone or in combination with vitamin B6.

A simple, sensitive and reproducible assay for pyridoxal 5'-phosphate and 4-pyridoxic acid in human plasma.

We describe a procedure for the measurement of pyridoxal 5'-phosphate and of 4-pyridoxic acid in human plasma samples. It is based on the conversion of pyridoxal 5'-phosphate to 4-pyridoxic acid 5'-phosphate by cyanide in alkaline medium, followed by a high pressure liquid chromatographic separation, with fluorescence detection at acid pH. The assay is robust, sensitive, linear over a wide range, reproducible, and simple to perform. Samples stored at -80 degrees C are stable. Satisfactory agreement was obtained with results from the tyrosine decarboxylase-based assay for pyridoxal 5'-phosphate, in two other laboratories. Plasma samples from a National Survey of older British people were analyzed, and reference intervals for plasma pyridoxal 5'-phosphate intervals were derived. From the lower 2.5 percentile of the reference group, taken as the lower cut-off of the normal range, ca. 20% of elderly men and 11% of elderly women in the UK showed evidence of biochemical deficiency.

An appraisal of vitamin B6 status indices and associated confounders, in young people aged 4-18 years and in people aged 65 years and over, in two national British surveys.

OBJECTIVE: To compare vitamin B6 status indices with each other and with potential confounding factors, in the datasets from two national British surveys and associated studies. DESIGN: Vitamin B6 status was measured by plasma pyridoxal phosphate (PLP) and plasma pyridoxic acid (PA) in both surveys, and also by erythrocyte aspartate aminotransferase activation coefficient (EAATAC) in one of the surveys. Plasma alpha1-antichymotrypsin was measured as an index of acute phase status; plasma creatinine was measured as an index of renal function; and plasma total alkaline phosphatase activity was measured as a proxy for PLP hydrolase activity. SETTING: The survey of people aged 65 years and over was carried out in 80 postcode sectors across mainland Britain during 1994-95 and the survey of young people was carried out in 132 postcode sectors across mainland Britain during 1997. SUBJECTS: Blood samples from c. 1,000 subjects of both sexes in each survey permitted measurements of plasma PLP and PA. There were also measurements of EAATAC in the young people's survey. RESULTS: According to published limits of normality, only 5% or less of the young people had unacceptable vitamin B6 status as measured by plasma PLP. About half had apparently unacceptable status by EAATAC, but this observation is difficult to interpret. The young people had considerably higher plasma concentrations of PLP and lower concentrations of PA than the older people. In both surveys, plasma PLP was strongly correlated with plasma PA and in the young persons' survey it was also correlated, although much less strongly, with the basal activity and activation coefficient of aspartate aminotransferase. Both plasma PLP and EAATAC (but not PA nor basal aspartate aminotransferase activity) were influenced by acute phase status in young people, as indicated by significant correlations with alpha1-antichymotrypsin. In people aged 65 years and over, PA (but not PLP) was correlated with renal function, as indicated by its relation with plasma creatinine; however PLP (but not PA) was correlated with plasma alkaline phosphatase activity. CONCLUSIONS: Several potential confounders - acute phase reaction, kidney malfunction and hydrolase activity - may influence vitamin B6 status indices, although differently for different indices and different age groups. Since older people have relatively poor vitamin B6 status, which may have important health implications for them, more reliable vitamin B6 status indices are needed.

North-south differences in some indices of Vitamin B6 nutritional status in older British people.

The National Diet and Nutrition Survey, nationally representative for the British population aged 65 years and over, has revealed a north-south geographical gradient, with a decline from south to north of vitamin B6 status indices. The present study further explores the possible explanatory factors (dietary intake of vitamin B6 and riboflavin, alcohol consumption, smoking habits and some other lifestyle determinants) on the difference of vitamin B6 indices--plasma concentrations of pyridoxal phosphate (pPLP) and pyridoxic acid (pPA), between older people living in the north (Scotland, North of England) and the south (Southern England, Wales and Midlands). The results showed that older people living in the northern half of Britain are at greater risk of poor vitamin B6 status, mainly as a result of low intakes of this vitamin, than the people living in the southern half of the country. Riboflavin intake, alcohol consumption, smoking and socio-economic status also correlated with the north-south gradient of pPLP and pPA. Other potential determinants such as use of vitamin B6 supplements, medicines probably affecting vitamin B6 metabolism, were not independent correlates of the north-south gradient in vitamin B6 status indices. This may have important implications for disease-risk geographical gradients in the UK.

Estimation of the use of dietary supplements in the National Diet and Nutrition Survey: people aged 65 years and Over. An observed paradox and a recommendation.

OBJECTIVE: To compare the evidence derived from blood biochemical status indices with the evidence from a questionnaire and from a 4-day weighed dietary record of micronutrient supplement use in the British National Diet and Nutrition Survey (NDNS) of People Aged 65 Years and Over; to resolve some apparent incompatibility between nutrient intake and status estimates, and to recommend an approach towards supplement recording that should improve accuracy. DESIGN: The survey procedures described in the National Diet and Nutrition Survey Report (1998) included a health-and-lifestyle questionnaire, a 4-day weighed diet record, and fasting blood and urine sample for biochemical indices, including a wide range of micronutrients. SETTING: Eighty randomly selected postcode sectors from mainland Britain during 1994-1995. SUBJECTS: Of 2060 people interviewed, 1467 provided a blood sample and 1217 provided both a blood sample, and a complete 4-day diet record. About 20% were living in institutions such as nursing homes, and the remainder were living in private households. RESULTS: After assigning the subjects to four categories by the use of dietary supplements (A, those not taking supplements (by questionnaire or by the 4-day record); B, those taking supplements (excluding prescribed ones) by questionnaire only; C, those taking supplements by 4-day record only; and D, those taking supplements by both questionnaire and 4-day record), these categories were then compared with respect to estimated total nutrient intakes and blood biochemical indices. Those in category B had estimated (4-day) nutrient intakes (from foods and supplements) that were indistinguishable from those in category A, but had biochemical indices that indicated significantly higher dietary intakes of several vitamins. CONCLUSIONS AND RECOMMENDATION: The 4-day weighed intake record may not have identified all of the subjects who were regularly taking micronutrient supplements in amounts sufficient to improve their biochemical status. Because survey respondents may use supplements irregularly or change their usual patterns of supplement use during a period of intensive diet-recording, it is important to design a dietary instrument that will minimise this potential source of inaccuracy. We therefore recommend that population surveys in which an accurate estimate of micronutrient intakes is required, from supplements as well as from food, should record supplement use for a period longer than 4-days. It is likely that a better estimate of long-term intakes can be achieved by combining a 4-day weighed diet record with a structured recall or several weeks of diary records, which focus specifically on the use of supplements.

[The lead level in the blood of pregnant women from the city of Sofia]. / Suderzhanie na olovo v kruvata na bremenni zheni ot grad Sofiia.

The study was aimed at establishing the relation between blood lead, nutrient intake and smoking habits of pregnant and nonpregnant women in Sofia. Forty four pregnant in the last trimester of pregnancy and 14 nonpregnant women were examined. There are no differences between the mean blood lead level of the groups. 22.72% of the pregnant and 37.7% of the nonpregnant women studied had blood lead above the accepted tolerable value of 0.48 mumol/l. It was established low intake of calcium, zinc and iron, minerals that contribute to lead bioavailability. A positive correlation between the number of cigarettes smoked and blood lead concentration was established. Based on this study it can be concluded that non-professional external exposure of lead in Sofia is significant. Smoking is eventually one contributing factor that can be excluded to reduce lead exposure.

[The evaluation of energy balance in pregnancy]. / Otsenka na energiiniia balans pri bremennost.

Energy balance assessment of 317 pregnant women in Sofia was conducted. Energy intake and energy expenditure during the first and the third trimester, gestational weight gain and prepregnancy body mass index have been studied. The regression analysis revealed a significant correlation between neonate body mass and energy expenditure level, but the decreased metabolic constants of physical activity, suggested an influence of increased basal metabolic rate, corresponding to the gestational body mass gain.

[Serum zinc and its relation to diet during late pregnancy]. / Serumen tsink i vruzkata mu s dietata po vreme na kusna bremennost.

The influence of dietary intake on serum zinc in 44 pregnant women in the third trimester of pregnancy and 10 healthy controls were studied. For assessment of the dietary intake 5-d record were used. Zinc in serum was analysed by flame AAS. Dietary zinc intake below the RDA was recorded in both groups studied (pregnant and nonpregnant women). Serum zinc in 42.8% of the pregnant women was below the cut off value. Only 1 of the nonpregnant women was with low serum zinc. For comparing serum zinc of both groups add ratio of 9.56 was calculated. Zinc in diet correlates significantly with the energy intake, protein and animal proteins in diet. Serum zinc correlates with animal protein intake and total energy of the diet. Pregnancy is a significant modulator of zinc status, but nutritional factor is a very important determinant for maintaining optimal nutritional zinc status.

[Changes in the level of lipid peroxidation in healthy pregnant women]. / Promeni v nivoto na lipidnata peroksidatsiia pri zdravi bremenni zheni.

A cross-sectional study on the level of lipid peroxidation and nutrient intake of 44 healthy pregnant women (34-36 gestation week) at average age 24.9 +/- 4.5 years and 15 controls (nonpregnant, nonlactating) at average age 31.8 +/- 7.6 years, was carried out. The level of lipid peroxidation was measured by the concentration of TBA-reactive substance (TBARS) in plasma and red blood cells and the concentration of reduced glutathione in red blood cells. The nutrient intake was studied by 5-day diet record. The results obtained showed an activation of lipid peroxidation in pregnant women evidenced by a statistically significant increase of TBARS in red blood cells. Nutrient density of the protein of animal origin, vitamin C and zinc was significantly diminished in the diet of pregnant women but their average daily intake of protein and vitamin C was higher than the recommended dietary allowances. Obviously the activated lipid peroxidation during pregnancy could not be related to inadequate intake of nutrients with antioxidant activity but most probably is a result of hormonal changes.

[The effect of the diet on the iron status and incidence of anemia in pregnant women]. / Vuzdeistvie na dietata vurkhu zhelezniia status i chestotata na anemiia pri bremenni zheni.

The influence of dietary intakes during I and III trimester of pregnancy on iron status and anemia incidence [correction of prevalence] in 44 healthy pregnant women were studied. For evaluation of iron intake the probability approach calculations were used. Iron status was assessed by using multiple criteria--iron in serum, TIBC, transferrin saturation capacity and hemoglobin level. Iron deficient anemia is diagnosed in pregnant woman when hemoglobin level is less than 110 g/l and the least two of the other indices are abnormal. More than 70% of the women studied have low dietary intake of iron during pregnancy, but only 21% are anemic. The half of women with anemia are with iron deficient anemia. The results of iron intake may overestimate the risk of developing iron deficiency. Iron status during pregnancy is influenced by the dietary intake and diet structure, iron body stores and adaptive mechanisms.