Modification of Gas6 Protein in the Brain by a Functional Endogenous Tissue Vitamin K Cycle.

The TAM receptor ligand Gas6 is known for regulating inflammatory and immune pathways in various organs including the brain. Gas6 becomes fully functional through the post-translational modification of multiple glutamic acid residues into γ-carboxyglutamic in a vitamin K-dependent manner. However, the significance of this mechanism in the brain is not known. We report here the endogenous expression of multiple components of the vitamin K cycle within the mouse brain at various ages as well as in distinct brain glial cells. The brain expression of all genes was increased in the postnatal ages, mirroring their profiles in the liver. In microglia, the proinflammatory agent lipopolysaccharide caused the downregulation of all key vitamin K cycle genes. A secreted Gas6 protein was detected in the medium of both mouse cerebellar slices and brain glial cell cultures. Furthermore, the endogenous Gas6 γ-carboxylation level was abolished through incubation with the vitamin K antagonist warfarin and could be restored through co-incubation with vitamin K1. Finally, the γ-carboxylation level of the Gas6 protein within the brains of warfarin-treated rats was found to be significantly reduced ex vivo compared to the control brains. In conclusion, we demonstrated for the first time the existence of a functional vitamin K cycle within rodent brains, which regulates the functional modification of endogenous brain Gas6. These results indicate that vitamin K is an important nutrient for the brain. Furthermore, the measurement of vitamin K-dependent Gas6 functionality could be an indicator of homeostatic or disease mechanisms in the brain, such as in neurological disorders where Gas6/TAM signalling is impaired.

Low vitamin K status in adults with cystic fibrosis is associated with reduced body mass index, insulin secretion, and increased pseudomonal colonization.

Patients with cystic fibrosis (CF) are at high risk of fat-soluble vitamin deficiencies, even with supplementation. The contribution of a suboptimal vitamin K status to respiratory and endocrine pathophysiology in CF has been inadequately characterized. This is a cross-sectional study in adult CF patients (≥18 years old) from the Montreal Cystic Fibrosis Cohort. Vitamin K1 (VK1) was measured with high-performance liquid chromatography, using fasted serum samples collected during an oral glucose tolerance test (OGTT: 2 h with plasma glucose and insulin every 30 min) (n = 168). Patients were categorized according to VK1 status (suboptimal defined as <0.30 nmol/L). Suboptimal VK1 levels were observed in 66% of patients. Patients with a suboptimal VK1 status have a higher risk of colonization with Pseudomonas aeruginosa (p = 0.001), have lower body mass index (BMI) (p = 0.003), and were more likely to have exocrine pancreatic insufficiency (p = 0.002). Using an established threshold for VK1, we did show significantly reduced OGTT-derived measures of insulin secretion in patients with a VK1 status below 0.30 nmol/L (first- and second-phase area under the curve (AUC)INS/GLU (p = 0.002 and p = 0.006), AUCINS (p = 0.012) and AUCINS/GLU (p = 0.004)). Subclinical vitamin K deficiency is more common than other fat-soluble vitamin deficiencies in patients with CF. We demonstrate an association between a suboptimal VK1 status and measures of insulin secretion. We highlight the potential associations of mild vitamin K deficiency with pseudomonal colonization and lower BMI, although these need to be validated in prospective studies.

Age-Related Increases in Marrow Fat Volumes have Regional Impacts on Bone Cell Numbers and Structure.

The increasing levels of bone marrow fat evident in aging and osteoporosis are associated with low bone mass and attributed to reduced osteoblastogenesis. Local lipotoxicity has been proposed as the primary mechanism driving this reduction in bone formation. However, no studies have examined the correlation between high levels of marrow fat volumes and changes in local cellularity. In this study, we hypothesize that areas of bone marrow with high fat volumes are associated with significant changes in cell number within a similar region of interest (ROI). Inbred albino Louvain (LOU) rats, originating from the Wistar strain, have been described as a model of healthy aging with the absence of obesity but expressing the typical features of age-related bone loss. We compared local changes in distal femur cellularity and structure in specific ROI of undecalcified bone sections from 4- and 20-month-old male and female LOU rats and Wistar controls. Our results confirmed that older LOU rats exhibited significantly higher fat volumes than Wistar rats (p < 0.001). These higher fat volume/total volume were associated with lower trabecular number (p < 0.05) and thickness (p < 0.05) and higher trabecular separation (p < 0.05). In addition, osteoblast and osteocyte numbers were reduced in the similar ROI containing high levels of adiposity, while osteoclast number was higher compared to control (p < 0.03). In summary, marrow ROIs with a high level of adiposity were associated with a lower bone mass and changes in cellularity explaining associated bone loss. Further studies assessing the levels of lipotoxicity in areas of high local marrow adiposity and identifying molecular actors involved in this phenomenon are still required.

Differential Effects of Long-Term Caloric Restriction and Dietary Protein Source on Bone and Marrow Fat of the Aging Rat.

Long-term caloric restriction (CR) has been shown to be beneficial to various tissues and organs. In contrast, CR exerts differential effects on bone, which could be due in part to the nature of the protein regime utilized. Male Sprague Dawley rats (8-month-old) were subjected for 12 months to 40% CR in macronutrients and compared with rats fed ad libitum for the same period. Casein- and soy-fed groups were compared. There was a significant decrease in bone quality in both CR groups, which was independent of the source of protein in the diet. In contrast, the group fed soy protein ad libitum showed better bone quality and higher levels of bone formation compared with casein-fed animals. Notably, bone marrow adipocytes were not mobilized upon CR as demonstrated by an absence of change in adipocyte number and tissue expression of leptin. This study demonstrates that the negative effect of CR on bone quality could not be prevented by the most common protein regimes.

Intracranial calcifications under vitamin K antagonists or direct oral anticoagulants: Results from the French VIKING study in older adults.

OBJECTIVES: The use of vitamin K antagonists (VKA) is associated with the onset of vascular and soft-tissue calcifications. Whether there are more intracranial calcifications under VKA remains unclear. The objective of this study was to determine whether the regular use of VKA in older adults was associated with an increased burden of intracranial calcifications compared with the use of direct oral anticoagulant (DOA). STUDY DESIGN: Nineteen patients aged 70 years or more using VKA for more than 3 months and 19 controls (matched for age, gender and indication for anticoagulation) using DOA for more than 3 months were consecutively included in this study. MAIN OUTCOMES MEASURES: The burden of intracranial calcifications was graded by an experienced neuroradiologist from 0 (no burden) to 3 (high burden) according to the quantity, size, intensity and confluence of calcifications on computed tomography scan of the brain. Age, gender, frontal assessment battery (FAB) score, hypertension, dyslipidaemia, carotid artery stenosis, kidney failure and indication for anticoagulation were investigated as potential confounders. RESULTS: The 19 patients using VKA (median[IQR], 84years[7]; 10females) exhibited a greater burden of falcian calcifications than the 19 controls using DOA (respectively, 2[1] versus 1[2], P = 0.025). Overall, we found that using VKA was directly associated with the global burden of intracranial calcifications (ß = 1.54, P = 0.049). No correlation was found with calcifications in sites other than the falx cerebri. CONCLUSIONS: The use of VKA was associated with a greater burden of intracranial calcifications compared with the use of DOA, specifically in the falx cerebri. This finding may explain part of the neurocognitive morbidity met with VKA.

Association between fat-soluble nutrient status and auditory and visual related potentials in newly diagnosed non-screened infants with cystic fibrosis: A case-control study.

Nutritional deficiencies often precede the diagnosis of cystic fibrosis (CF) in infants, and occur at a stage where the rapidly developing brain is more vulnerable to insult. We aim to compare fat-soluble nutrient status of newly diagnosed non-screened infants with CF to that of healthy infants, and explore the association with neurodevelopment evaluated by electroencephalography (EEG). Our results show that CF infants had lower levels of all fat-soluble vitamins and docosahexaenoic acid (DHA) compared to controls. The auditory evoked potential responses were higher in CF compared to controls whereas the visual components did not differ between groups. DHA levels were correlated with auditory evoked potential responses. Although resting state frequency power was similar between groups, we observed a negative correlation between DHA levels and low frequencies. This study emphasizes the need for long-term neurodevelopmental follow-up of CF infants and pursuing intervention strategies in the future.

Brain region-specific effects of long-term caloric restriction on redox balance of the aging rat.

Caloric restriction (CR) is the most effective intervention to improve health span and extend lifespan in preclinical models. This anti-aging effect of CR is related to attenuation of oxidative damage in various tissues, with divergent results in the brain. We addressed how brain oxidoreductive balance would be modulated in male Sprague-Dawley (SD) rats submitted to a 40% CR from 8 to 19 months of age, by reference to ad libitum-fed (AL) rats at 2 and 19 months of age. Four brain structures were compared: hippocampus, striatum, parietal cortex, cerebellum. Our CR diet elicits significant prevention of oxidative damages with the upregulation of antioxidant defenses (levels of glutathione [GSH], mRNAs of clusterin and of three key antioxidant enzymes) as compared to age-matched AL controls, in a strikingly region-specific pattern. CR also prevented a drastic rise of the glial fibrillary acidic protein in the hippocampus of old AL rats. Besides, the CR effects at age 19 months mainly consist in improving endogenous defenses before the onset of age-related redox alterations. These effects are more prominent in the hippocampus.

Gender- and region-dependent changes of redox biomarkers in the brain of successfully aging LOU/C rats.

The LOU/C (LOU) rat is an obesity resistant strain with higher longevity and healthspan than common rats. The management of oxidative stress being important to successful aging, we characterized this process in the aging LOU rat. Male/female LOU rats were euthanized at 4, 20, and 29 months. Macrodissected hippocampus, striatum, parietal cortex, cerebellum were assayed for tissue concentrations of glutathione (GSH), gamma-glutamyl-cysteine-synthetase (γ-GCS), total thiols, protein carbonyls, mRNAs of clusterin and the known protective enzymes thioredoxine-1 (TRX-1), glutaredoxine-1 (GLRX-1), superoxide dismutase-1 (SOD-1). Brain levels of GSH, γ-GCS, total thiols remained constant with age, except for GSH and γ-GCS which decreases in females. Clusterin, TRX-1, GLRX-1, SOD-1 mRNA levels were maintained or increased in the hippocampus with age. Age-dependency of the markers differed between sexes, with SOD-1 and TRX-1 decreases out of hippocampus in females. Since antioxidants were reported to decrease with age in the brain of Wistar rats, maintenance of GSH levels and of protective enzymes mRNA levels in the LOU rat brain could contribute to the preservation of cognitive functions in old age. Altogether, the successful aging of LOU rats may, at least in part, involve the conservation of functional antioxidant mechanisms in the brain, supporting the oxidative stress theory of aging.

Aging and diets regulate the rat anterior pituitary and hypothalamic transcriptome.

Dietary interventions involving caloric restriction represent a powerful strategy to prevent or delay age-related deteriorations and diseases. Their beneficial effects have been observed in several tissues and species. This microarray study investigated the effects of aging, long-term moderate caloric restriction (LTMCR) and long-term dietary soy on the regulation of gene expression in the anterior pituitary and hypothalamus of 20-month-old Sprague-Dawley rats. In both tissues, aging regulated genes mainly involved in cell defense and repair mechanisms related to apoptosis, DNA repair, cellular stress, inflammatory and immune response. In the aging pituitary, the highest upregulated gene was the regenerating islet-derived 3ß (5.77-fold), coding for a secretory protein involved in acute stress and inflammation. A protective effect of LTMCR on age-related change of gene expression was observed for 35 pituitary genes. In addition, beneficial effects of LTMCR in the pituitary were observed on new regulated genes mainly involved in cell death and cell stress response. In the hypothalamus, the effects of LTMCR on age-related changes were modest. Finally, changing the quality of dietary protein (20% casein for soy) had a low impact on the regulation of mRNA levels in both tissues. Genes associated with the somatotroph function were also differentially expressed in the aging pituitary. Interestingly, LTMCR prevented the effect of aging on insulin-like growth factor-binding protein-3 gene. Altogether, this study proposes novel pituitary and hypothalamic molecular targets and signaling pathways to help in understanding the mechanisms involved in aging processes and LTMCR.

Sodium intake and physical activity impact cognitive maintenance in older adults: the NuAge Study.

This study examines the association between sodium intake and its interaction with physical activity on cognitive function over 3 years in older adults residing in Québec, Canada. We analyzed a subgroup from the NuAge cohort (aged 67-84 years) with nutrient intake data, including sodium, from a food frequency questionnaire administered at baseline. Baseline physical activity was assessed using the Physical Activity Scale for the Elderly (PASE; high-low). Modified Mini Mental State Examination (3MS) was administered at baseline and annually for 3 additional years. Controlling for age, sex, education, waist circumference, diabetes, and dietary intakes, analyses showed an association between sodium intake and cognitive change over time in the low PASE group only. Specifically, in the low PASE group, elders in the low sodium intake tertile displayed better cognitive performance over time (mean decline in 3MS over years: mean [M] = -0.57, standard error [SE] = 0.002) compared with the highest (M = -1.72, SE = 0.01) and mid sodium intake (M = -2.07, SE = 0.01) groups. This finding may have significant public health implications, emphasizing the importance of addressing multiple lifestyle factors rather than a single domain effect on brain health.

Drugs, nutrients, and phytoactive principles improving the health span of rodent models of human age-related diseases.

Rodents are often the species of choice to examine the effect of drugs on survival and on the progression of specific diseased tissues. This statement is also true for research laboratories working in the field of nutrition and aging. In addition to diets that can reduce the life expectancy of rodents, such as diabetogenic or high-fat diets, genetically modified rodents exhibiting different accelerated age-associated diseases also provide important biologic tools to decipher the impact of drugs, nutrients, or phytoactive compounds on their health and life span. This review covers some of the chemicals believed to decelerate the appearance of age-related diseases in different rodent models. Such chemicals include antioxidants, anti-inflammatory molecules, modulators of metabolic sensors, calorie restriction mimetics, and vegetal polyphenolic compounds that affect mitochondrial functions, cellular proliferation or differentiation as well as cell functionality.

Catechin treatment improves cerebrovascular flow-mediated dilation and learning abilities in atherosclerotic mice.

Severe dyslipidemia and the associated oxidative stress could accelerate the age-related decline in cerebrovascular endothelial function and cerebral blood flow (CBF), leading to neuronal loss and impaired learning abilities. We hypothesized that a chronic treatment with the polyphenol catechin would prevent endothelial dysfunction, maintain CBF responses, and protect learning abilities in atherosclerotic (ATX) mice. We treated ATX (C57Bl/6-LDLR(-/-)hApoB(+/+); 3 mo old) mice with catechin (30 mg · kg(-1) · day(-1)) for 3 mo, and C57Bl/6 [wild type (WT), 3 and 6 mo old] mice were used as controls. ACh- and flow-mediated dilations (FMD) were recorded in pressurized cerebral arteries. Basal CBF and increases in CBF induced by whisker stimulation were measured by optical coherence tomography and Doppler, respectively. Learning capacities were evaluated with the Morris water maze test. Compared with 6-mo-old WT mice, cerebral arteries from 6-mo-old ATX mice displayed a higher myogenic tone, lower responses to ACh and FMD, and were insensitive to NOS inhibition (P < 0.05), suggesting endothelial dysfunction. Basal and increases in CBF were lower in 6-mo-old ATX than WT mice (P < 0.05). A decline in the learning capabilities was also observed in ATX mice (P < 0.05). Catechin 1) reduced cerebral superoxide staining (P < 0.05) in ATX mice, 2) restored endothelial function by reducing myogenic tone, improving ACh- and FMD and restoring the sensitivity to nitric oxide synthase inhibition (P < 0.05), 3) increased the changes in CBF during stimulation but not basal CBF, and 4) prevented the decline in learning abilities (P < 0.05). In conclusion, catechin treatment of ATX mice prevents cerebrovascular dysfunctions and the associated decline in learning capacities.

Effects of long-term dietary interventions on pituitary growth hormone-releasing hormone receptor in aging rats and potential mechanisms of action.

Long-term moderate caloric restriction (LTMCR) is a powerful intervention to delay age-related health deterioration. In this study, the effects of 40% caloric restriction (CR), implemented at 8 months with or without protein restriction, and 40% dietary protein restriction alone, implemented at 2 months, were examined on pituitary growth hormone-releasing hormone receptor (GHRH-R) and GHRH sensitivity in 12-20-month-old male Sprague Dawley rats. An increase of the 4-kb GHRH-R mRNA transcript and 4 kb/2.5 kb ratio, the blunting of high affinity GHRH binding sites and a decrease in GHRH-induced cAMP production were observed in old rats. Only 40% CR maintained youthful levels of GHRH-R transcripts and GHRH binding parameters as well as maximal GHRH-induced cAMP production. Moreover, the GHRH-induced capacity of somatotrophs to synthesize/repair DNA in vitro, in the presence of moderate glucotoxic stress (12 mM d-glucose), was maintained in old CR rats. Among the hormonal and metabolic regulators of GHRH-R that were modified by LTMCR, glucose, free fatty acids and glucocorticoids represent promising candidates for future investigation. A better understanding of the molecular and cellular mechanisms by which they regulate the pituitary GHRH-R should help define strategies to mimic the beneficial effects of LTMCR by less demanding interventions.

Age-related bone loss in the LOU/c rat model of healthy ageing.

Inbred albino Louvain (LOU) rats are considered a model of healthy aging due to their increased longevity in the absence of obesity and with a low incidence of common age-related diseases. In this study, we characterized the bone phenotype of male and female LOU rats at 4, 20 and 27 months of age using quantitative micro computed tomographic (mCT) imaging, histology and biochemical analysis of circulating bone biomarkers. Bone quality and morphometry of the distal femora, assessed by mCT, was similar in male and female rats at 4 months of age and deteriorated over time. Histochemical staining of undecalcified bone showed a significant reduction in cortical and trabecular bone by 20 months of age. The reduction in mineralized tissue was accompanied by reduced numbers of osteoblasts and osteoclasts and a significant increase in marrow adiposity. Biochemical markers of bone turnover, C-telopeptide and osteocalcin, correlated with the age-related bone loss whereas the calciotropic hormones PTH and vitamin D remained unchanged over time. In summary, aged LOU rats exhibit low-turnover bone loss and marrow fat infiltration, which are the hallmarks of senile osteoporosis, and thus represent a novel model in which to study the molecular mechanisms leading to this disorder.

Prevalence of low bone mass and deficiencies of vitamins D and K in pediatric patients with cystic fibrosis from 3 Canadian centers.

OBJECTIVE: In this cross-sectional observational study, we assessed both vitamins D and K status and bone health in pancreatic insufficient pediatric patients with cystic fibrosis from 3 Canadian cystic fibrosis centers. METHODS: Eighty-one patients who had cystic fibrosis and were clinically stable for at least 3 months were enrolled. At the time of the clinic visit, anthropometric variables, lung function, pubertal status, intake of calcium and vitamins D and K, and physical activity were assessed. Blood was taken for analysis of biochemical biomarkers of bone turnover and status of vitamins D and K, and a urine sample was obtained for calcium, creatinine, sodium, and deoxypyridoline analyses. Whole-body bone mineral content and lumbar spine (L1-L4) bone mineral density were measured. RESULTS: The children were relatively well nourished and had moderate to mild lung disease. Low bone mineral mass defined as a z score between -1.0 and -2.0, for gender and age was detected in 38% of the children for whole body and in 28% for lumbar spine. z score less than -2.0 was observed in 7 children for both bone measures. Suboptimal vitamin D status occurred in 95% of patients; suboptimal vitamin K status occurred in 82% of patients. Measures of plasma osteocalcin and carboxy-terminal propeptide type 1 procollagen and urinary deoxypyridoline compared with reference values for age, gender, and pubertal status reflected a state of suppressed bone formation and elevated bone resorption in a large proportion of the patients. CONCLUSIONS: Bone mass of the whole body and spine was lower than expected for chronological age in approximately one third of pediatric patients with cystic fibrosis irrespective of gender or age. This may be explained by the observation of low bone turnover for developmental stage as indicated by bone biomarkers. Suboptimal status of vitamins D and K may be key causative factors of the low bone status for age.

Efficacy of high dose phylloquinone in correcting vitamin K deficiency in cystic fibrosis.

BACKGROUND: Subclinical deficiencies of vitamin K are universally present in unsupplemented cystic fibrosis (CF) patients. The dose required to prevent deficiencies cannot be estimated from the existing literature. The aim of this study is determine if a supplemental dose of 1 mg/day or 5 mg/day vitamin K1 per day would normalize vitamin K status in a population of children with cystic fibrosis. METHODS: Fourteen pancreatic insufficient CF children, between the ages of 8 to 18 years old, were randomized to receive either 1 mg/day or 5 mg/day vitamin K1 per day, for one month. Fasting blood tests were done at baseline and after one month of the intervention. The degree of undercarboxylation of osteocalcin (%Glu-OC), and serum vitamin K1, were evaluated by descriptive statistics and nonparametric Wilcoxon matched-pair test and Mann-Whitney U test. RESULTS: Of the 50% of subjects who were below the optimal serum vitamin K1 at baseline, all rose into the normal range with supplementation. Supplementation also significantly reduced the overall %Glu-OC from a median of 46.8 to 29.1% (p<0.0003). CONCLUSION: Our results suggest that both 1 mg and 5 mg of vitamin K1, given over a one-month period in pancreatic insufficient pediatric cystic fibrosis patients improve vitamin K status.

Diet quality of Montreal-area adults needs improvement: estimates from a self-administered food frequency questionnaire furnishing a dietary indicator score.

OBJECTIVE/DESIGN: Time and budget constraints limit nutrition evaluation in Canadian health surveys. To encourage regular population diet monitoring in Canada, we developed and assessed the relative validity of a population-based food frequency questionnaire (FFQ) to assess usual diet in Quebecers. A 73-item, self-administered semiquantitative FFQ was designed in French and English from the Block National Cancer Institute Health Habits and History Questionnaire to capture usual food consumption among Quebec adults. The US Department of Agriculture Healthy Eating Index (HEI) (maximum score=100) was adapted to Canadian age- and sex-specific dietary recommendations, validated, and programmed to score the FFQ for diet quality. SUBJECTS: The FFQ was pretested, pilot tested, and administered by mail to a random sample of 248 adults aged 18 to 82 years in the Montreal area (57% women) who were recruited by random digit dialing (64% response rate). A subgroup (n=94) furnished four nonconsecutive 1-day food records for validation of the FFQ. RESULTS: Mean FFQ energy intakes were 2,216 kcal (median 2,110 kcal) for men and 1,785 kcal (median 1,680 kcal) for women. Proportional median macronutrient consumption was similar in both sexes at 17% protein, 34% to 35% fats, and 48% to 49% carbohydrates, but differed by age group in women. Adequacy of micronutrient intakes relative to Dietary Reference Intakes varied by age, sex, and nutrient. Whereas most mean or median intakes were at or exceeded recommendations, calcium intakes were low overall and levels of several other nutrients were very low at the 10th percentile. Mean Canadian HEI (range 46 to 99) was higher in women (74.9) than men (70.3, P<.001). Women's scores showed they met recommendations for intakes of vegetables and fruit, cholesterol, and sodium better than men (P ranged from <.01 to <.0001), while men fared better at meeting recommended fat intake levels (P<.05). CONCLUSIONS: The Canadian HEI adequately discriminates overall diet quality based on dietary data estimated from our FFQ. Examination of subscores within and between quartiles may best reveal which food groups require attention to improve diet quality, providing valuable information for teaching and planning. Future studies should test diet quality indicators in populations recruited to reflect greater dietary diversity and reporting ability and include members of disadvantaged groups to provide a broader set of behaviors that could shed light on factors influencing diet quality.

Development and validation of a food frequency questionnaire.

Regular diet monitoring requires a tool validated in the target population. A 73-item, semiquantitative, self-administered food frequency questionnaire (FFQ), was adapted in French and English from the Block National Cancer Institute Health Habits and History Questionnaire. The FFQ was used to capture usual long-term food consumption among adults living in Quebec. A representative sample of adults aged 18 to 82 (57% female) was recruited by random digit dialling in the Montreal region. Approximately 64% of recruits completed and returned the instrument by mail (n=248). The FFQ was validated in a subsample (n=94, 61% female) using four nonconsecutive food records (FRs). Median energy intakes (in kcal) for men and women, respectively, were FFQ (total sample) 2,112 and 1,823, FFQ (subsample) 2,137 and 1,752, and FR (subsample) 2,510 and 1,830. Spearman correlation analyses between FFQ and FR nutrients were positive (with r ranging from 0.32 for folate to 0.58 for saturated fatty acids) and statistically significant (p<0.001), with better results among women. On average, cross-classification of energy and 24 nutrients from the FFQ and means of four FRs placed 39% into identical quartiles and 78% into identical and contiguous quartiles, with only 4% frankly misclassified. These results suggest that the FFQ is a relatively valid instrument for determining usual diet in Quebec adults.