Bioactivity and Bioavailability of Carotenoids Applied in Human Health: Technological Advances and Innovation.

This article presents a groundbreaking perspective on carotenoids, focusing on their innovative applications and transformative potential in human health and medicine. Research jointly delves deeper into the bioactivity and bioavailability of carotenoids, revealing therapeutic uses and technological advances that have the potential to revolutionize medical treatments. We explore pioneering therapeutic applications in which carotenoids are used to treat chronic diseases such as cancer, cardiovascular disease, and age-related macular degeneration, offering novel protective mechanisms and innovative therapeutic benefits. Our study also shows cutting-edge technological innovations in carotenoid extraction and bioavailability, including the development of supramolecular carriers and advanced nanotechnology, which dramatically improve the absorption and efficacy of these compounds. These technological advances not only ensure consistent quality but also tailor carotenoid therapies to each patient's health needs, paving the way for personalized medicine. By integrating the latest scientific discoveries and innovative techniques, this research provides a prospective perspective on the clinical applications of carotenoids, establishing a new benchmark for future studies in this field. Our findings underscore the importance of optimizing carotenoid extraction, administration, bioactivity, and bioavailability methods to develop more effective, targeted, and personalized treatments, thus offering visionary insight into their potential in modern medical practices.

Bioaccessibility and Cellular Uptake of Carotenoids Extracted from Bactris gasipaes Fruit: Differences between Conventional and Ionic Liquid-Mediated Extraction.

Currently, on an industrial scale, synthetic colorants are used in many fields, as well as those extracted with conventional organic solvents (COSs), leading to several environmental issues. Therefore, we developed a sustainable extraction and purification method mediated by ionic liquids (IL), which is considered an alternative high-performance replacement for COSs. Carotenoids are natural pigments with low bioaccessibility (BCT) and bioavailability (BV) but with huge importance to health. To investigate if the BCT and cellular uptake of the carotenoids are modified by the extraction method, we conducted a comparison assay between both extraction procedures (IL vs. COS). For this, we used the Amazonian fruit Bactris gasipaes, a rich source of pro-vitamin A carotenoids, to obtain the extract, which was emulsified and subjected to an in vitro digestion model followed by the Caco-2 cell absorption assay. The bioaccessibility of carotenoids using IL was better than those using COS (33.25%, and 26.84%, respectively). The cellular uptake of the carotenoids extracted with IL was 1.4-fold higher than those extracted using COS. Thus, IL may be a feasible alternative as extraction solvent in the food industry, replacing COS, since, in this study, no IL was present in the final extract.

Modified-release of encapsulated bioactive compounds from annatto seeds produced by optimized ionic gelation techniques.

To compare the encapsulation of annatto extract by external gelation (EG) and internal gelation (IG) and to maximize process yield (% Y), two central composite designs were proposed. Calcium chloride (CaCl2) concentration (0.3-3.5%), alginate to gelling solution ratio (1:2-1:6); acetic acid (CH3COOH) concentration (0.2-5.0%) and alginate to gelling solution ratio (1:2-1:6) were taken as independent variables for EG and IG respectively. Release studies were conducted under different conditions; morphology, particle size, the encapsulation efficiency (EE), and release mechanism were evaluated under optimized conditions. The optimized EG conditions were 0.3% CaCl2 and 1:1.2 alginate to gelling solution ratio, whereas a 0.3% CH3COOH and 1:5 alginate to gelling solution ratio were optimized conditions for IG. When 20% extract was employed, the highest EE was achieved, and the largest release was obtained at a pH 6.5 buffer. The Peppas-Sahlin model presented the best fit to experimental data. Polyphenol release was driven by diffusion, whereas bixin showed anomalous release. These results are promising for application as modulated release agents in food matrices.

Bioaccessibility and cellular uptake by Caco-2 cells of carotenoids and chlorophylls from orange peels: A comparison between conventional and ionic liquid mediated extractions.

Native extracts from orange peels were obtained by a conventional method using acetone and, an alternative method using ionic liquid (1-butyl-3-methylimidazolium chloride ([C4mim]Cl)). The bioaccessibilities and cellular uptakes of carotenoids, esters and chlorophylls were evaluated, since the influence of esterification on bioaccessibility and bioavailability is not well established. For this, the extracts were emulsified, submitted to in vitro simulated digestion model according to the INFOGEST protocol, followed by uptake by Caco-2 cells. Compounds were separated, identified and quantified by HPLC-PDA-MS/MS. After digestion, 22.0% and 26.2% of the total carotenoids and 45.9% and 68.7% of the chlorophylls were bioaccessible from the acetone and [C4mim]Cl extracts, respectively. The bioaccessibilities of xanthophylls and carotenes were significantly higher than those of the mono- and diesters. The uptake by Caco-2 cells varied from 130.2 to 131.9 ng/mg cell protein for total carotenoids and from 243.8 to 234.2 ng/mg cell protein for chlorophylls in the acetone and [C4mim]Cl extracts, respectively. In general, xanthophylls and esters were better absorbed than carotenes.

Dietary Carotenoid Roles in Redox Homeostasis and Human Health.

Classic nutrition believed that healthy diets should simply provide sufficient antioxidant loads to organisms, to hamper free radical processes and avoid oxidative stress. Current redox biology was proven much more intricate. Carotenoids are bioactive compounds in the human diet with a multifaceted role in redox metabolism. This perspective discusses the participation of α/ß-carotene, lutein, zeaxanthin, lycopene, ß-cryptoxanthin, astaxanthin, and derivatives in redox homeostasis focusing on (i) their antioxidant/pro-oxidant activities, (ii) control of gene expression via Nrf2-Keap1 and NF-κB pathways, and (iii) their link with (sub)cellular redox circuits, as part of the "redox code" that orchestrates physiological processes and health in humans.

Lycopene-rich tomato oleoresin modulates plasma adiponectin concentration and mRNA levels of adiponectin, SIRT1, and FoxO1 in adipose tissue of obese rats.

AIM: To investigate whether lycopene can modulate adiponectin levels and SIRT1 and FoxO1 gene expression in the adipose tissue of diet-induced obese rats. METHODS: Male Wistar rats were first fed with hypercaloric diet (HD, n = 12) for 6 weeks, and afterward, these rats were randomly assigned to receive HD (n = 6) or HD with lycopene-rich tomato oleoresin (equivalent to lycopene 10 mg/kg body weight (BW)/day, HD + L, n = 6) by gavage for additional 6 weeks. Plasma lycopene and adiponectin levels were analyzed by high-performance liquid chromatography and immunoassay, respectively. The messenger RNA (mRNA) expressions of adiponectin, Sirtuin 1 (SIRT1), Forkhead box O 1 (FoxO1), fatty acid translocase/cluster of differentiation 36 (FAT/CD36), and PPARγ in adipose tissues were determined by quantitative polymerase chain reaction. RESULTS: Lycopene was detected in the plasma of rats in HD + L group but not in the HD group. Although both BW and adiposity were not different between the two groups, there was a significant increase in both plasma concentration and mRNA expression of adiponectin in the adipose tissue of the HD + L group. In addition, the lycopene supplementation upregulated mRNA expressions of SIRT1, FoxO1, and FAT/CD36 but downregulated PPARγ in adipose tissue of obese rats. CONCLUSION: These data suggest that lycopene, in the concentration used, is not toxic and also its health benefits in adipose tissue may play a role against obesity-related complications.

Retention of provitamin a carotenoids in staple crops targeted for biofortification in Africa: cassava, maize and sweet potato.

HarvestPlus, part of the Consultative Group on Internation Agriculture research (CGIAR) Program on Agriculture for Nutrition and Health (A4NH) uses conventional plant breeding techniques to develop staple food crops that are rich in micronutrients, a food-based approach to reduce micronutrient malnutrition known as biofortification. The nutritional breeding targets are established based on the food intake of target populations, nutrient losses during storage and processing and bioavailability. This review collates the evidence on the retention of provitamin A carotenoid (pVAC) after processing, cooking, and storing of the staple crops targeted for pVAC biofortification: cassava, maize, and sweet potato. Sun drying was more detrimental to the pVAC levels (27-56% retention) in cassava than shade (59%) or oven (55-91%) drying, while the pVAC retention levels (66-96%) in sweet potato were not significantly different among the various drying methods. Overall, boiling and steaming had higher pVAC retention (80-98%) compared to baking (30-70%) and frying (18-54%). Gari, the most frequently consumed form of cassava in West Africa had the lowest pVAC retention (10-30%). The pVAC retention of maize grain and cassava and sweet potato flour reached levels as low as 20% after 1-4 months of storage and was highly dependent on genotype. Therefore, we recommend that an evaluation of the pVAC degradation rate among different genotypes be performed before a high pVAC crop is promoted.

Effects of carotenoid and vitamin E supplementation on oxidative stress and plumage coloration in house finches (Haemorhous mexicanus).

There has been much recent interest from both applied and basic scientists in the broad series of benefits that animals reap from acquiring high concentrations of dietary antioxidants, such as carotenoids and vitamins (e.g., vitamin E, or tocopherol). Most attention has been paid to separate effects of these compounds on, for example, coloration, health state, development, and vision, but because of possible interactions between these lipid-soluble molecules, we are in need of more studies that co-manipulate these substances and examine their possible synergistic impacts on animal physiology and phenotype. We capitalized on a model avian system (the house finch, Haemorhous mexicanus), where extensive information is available on the fitness roles of carotenoids, to test how variation in carotenoid and/or vitamin E concentrations in the diet impacts body accumulation of these compounds, factors related to oxidative damage (e.g., breast muscle and plasma oxidative-stress susceptibility, plasma nitric-oxide levels), and plumage color development. As in a previous study of ours on carotenoids and health in finches, we employed a 2×2 factorial experimental design on birds in both molting and non-molting conditions, to understand how seasonal shifts in carotenoid use (i.e., pigment incorporation into plumage) might alter the accumulation and roles of carotenoids and vitamins. As expected, lutein supplementation increased the level of circulating carotenoids in both experiments and the color of newly molted plumage. By contrast, vitamin E provisioning did not significantly affect plasma carotenoid levels or plumage coloration in either experiment. Interestingly, carotenoid provisioning decreased circulating vitamin E levels during molt, which suggests either molecular competition between carotenoids and tocopherol at the absorption/transport stages or that vitamin E serves as an antioxidant to offset harmful actions that carotenoids may have at very high concentrations. Finally, in both experiments, we found a reduction in breast-muscle oxidative damage for tocopherol-supplemented birds, which constitutes the first demonstration of a protective effect of vitamin E against oxidative stress in wild birds. Taken together, these findings provide an interesting contrast with our earlier work on season-specific physiological benefits of carotenoids in finches and point to complex associations between indicators of antioxidant and oxidative state in wild-caught animals.

Effect of ditaxin and heteranthin and inhibitory effect of Ditaxis heterantha extract on L5178Y tumor development in mice.

Ditaxis heterantha seeds are used as spices for flavoring and coloring food. Two new apocarotenoids derived from the seeds, heteranthin and ditaxin, were evaluated for their in vitro cytotoxic effects in murine lymphoma cells lines. Bioabsorption in mice and preventive and antitumor effects of the apocarotenoids were determined. Ditaxin and heteranthin showed cytotoxic effects in vitro against murine malignant cells and normal splenocyte cells. The 50% inhibitory concentration (IC(50)) for ditaxin in splenocytes was 0.1825 mM; in L5178Y, the IC(50) was 0.1923 mM. The heteranthin IC(50) in splenocytes was 0.1325 mM; in L5178Y, the value was 0.3889 mM. The maximum ditaxin plasma concentration was found after 2 hours of administration (mean±standard deviation, 7.5±2.05 µg/mL). Oral administration of the D. heterantha extract (100 mg/kg per day) for 14 days after the L5178Y lymphoma cell implantation showed no significant effect compared with groups that were not pretreated. However, tumor inhibition in groups treated intraperitoneally before inoculation with the L5178Y cells showed a significant difference (P<.001) compared with the groups not pretreated.

Doxorubicin as an antioxidant: maintenance of myocardial levels of lycopene under doxorubicin treatment.

The mechanism of doxorubicin-induced cardiotoxicity remains controversial. Wistar rats (n=96) were randomly assigned to a control (C), lycopene (L), doxorubicin (D), or doxorubicin+lycopene (DL) group. The L and DL groups received lycopene (5 mg/kg body wt/day by gavage) for 7 weeks. The D and DL groups received doxorubicin (4 mg/kg body wt intraperitoneally) at 3, 4, 5, and 6 weeks and were killed at 7 weeks for analyses. Myocardial tissue lycopene levels and total antioxidant performance (TAP) were analyzed by HPLC and fluorometry, respectively. Lycopene metabolism was determined by incubating (2)H(10)-lycopene with intestinal mucosa postmitochondrial fraction and lipoxygenase and analyzed with HPLC and APCI mass spectroscopy. Myocardial tissue lycopene levels in DL and L were similar. TAP adjusted for tissue protein were higher in myocardium of D than those of C (P=0.002). Lycopene metabolism study identified a lower oxidative cleavage of lycopene in D as compared to those of C. Our results showed that lycopene was not depleted in myocardium of lycopene-supplemented rats treated with doxorubicin and that higher antioxidant capacity in myocardium and less oxidative cleavage of lycopene in intestinal mucosa of doxorubicin-treated rats suggest an antioxidant role of doxorubicin rather than acting as a prooxidant.

Effect of lycopene on doxorubicin-induced cardiotoxicity: an echocardiographic, histological and morphometrical assessment.

Doxorubicin is an excellent chemotherapeutic agent utilized for several types of cancer but the irreversible doxorubicin-induced cardiac damage is the major limitation for its use. Oxidative stress seems to be associated with some phase of the toxicity mechanism process. To determine if lycopene protects against doxorubicin-induced cardiotoxicity, male Wistar rats were randomly assigned either to control, lycopene, doxorubicin or doxorubicin + lycopene groups. They received corn oil (control, doxorubicin) or lycopene (5 mg/kg body weight a day) (lycopene, doxorubicin + lycopene) by gavage for a 7-week period. They also received saline (control, lycopene) or doxorubicin (4 mg/kg) (doxorubicin, doxorubin + lycopene) intraperitoneally by week 3, 4, 5 and 6. Animals underwent echocardiogram and were killed for tissue analyses by week 7. Mean lycopene levels (nmol/kg) in liver were higher in the doxorubicin + lycopene group (5822.59) than in the lycopene group (2496.73), but no differences in lycopene were found in heart or plasma of these two groups. Lycopene did not prevent left ventricular systolic dysfunction induced by doxorubicin. However, morphologic examination revealed that doxorubicin-induced myocyte damage was significantly suppressed in rats treated with lycopene. Doxorubicin treatment was followed by increase of myocardium interstitial collagen volume fraction. Our results show that: (i) doxorubicin-induced cardiotoxicity was confirmed by echocardiogram and morphological evaluations; (ii) lycopene absorption was confirmed by its levels in heart, liver and plasma; (iii) lycopene supplementation provided myocyte protection without preventing interstitial collagen accumulation increase; (iv) doxorubicin-induced cardiac dysfunction was not prevented by lycopene supplementation; and (v) lycopene depletion was not observed in plasma and tissues from animals treated with doxorubicin.

Carotenoids from malanga (Xanthosoma sagittifolium) leaves protect cells against oxidative stress in rats.

The present study investigated the effect of malanga leaf powder and its carotenoids oil extract on oxidative damage in rat tissues. A group of rats received AIN-93G diet devoid of vitamin A (AD) and three other groups were treated with AD diet supplied with synthetic beta-carotene (beta-car) or malanga leaf powder (MP) or malanga carotenoids extract (CE). The diets' carotenoids bioavailability was determined through carotenoids intake/liver retinol content. Lipid thiobarbituric acid reactive substances (TBARS) and protein (carbonyl) oxidation, and reduced (GSH) and oxidized (GSSG) glutathione concentrations were determined in liver, heart, and brain. Synthetic beta-carotene bioavailability was higher than that of pro-vitamin A carotenoids from MP and CE diets, and no difference was observed between the latter two groups. Liver and heart lipid peroxidation was lower in the beta-car and CE groups than the AD group, while no difference was observed for the MP group. The beta-car, MP, and CE groups showed lower liver protein oxidation than the AD group, and only the CE group had lower heart protein oxidation in relation to AD. The MP group had a lower liver GSSG concentration and higher GSH/GSSG ratio than the AD group, while no difference was observed for heart glutathione concentration among the groups. The results indicate that at physiological levels, beta-car, malanga carotenoids extract, and malanga leaf powder have antioxidant effects in rats.

Food carotenoids: analysis, composition and alterations during storage and processing of foods.

Substantial progress has been achieved in recent years in refining the analytical methods and evaluating the accuracy of carotenoid data. Although carotenoid analysis is inherently difficult and continues to be error prone, more complete and reliable data are now available. Rather than expressing the analytical results as retinol equivalents, there is a tendency to present the concentrations of individual carotenoids, particularly beta-carotene, beta-cryptoxanthin, alpha-carotene, lycopene, lutein and zeaxanthin, carotenoids found in the human plasma and considered to be important to human health in terms of the provitamin A activity and/or reduction of the risk for developing degenerative diseases. With the considerable effort directed to carotenoid analysis, many food sources have now been analyzed in different countries. The carotenoid composition of foods vary qualitatively and quantitatively. Even in a given food, compositional variability occurs because of factors such as stage of maturity, variety or cultivar, climate or season, part of the plant consumed, production practices, post-harvest handling, processing and storage of food. During processing, isomerization of trans-carotenoids, the usual configuration in nature, to the cis-forms occurs, with consequent alteration of the carotenoids' bioavailability and biological activity. Isomerization is promoted by light, heat and acids. The principal cause of carotenoid loss during processing and storage of food is enzymatic or non-enzymatic oxidation of the highly unsaturated carotenoid molecules. The occurrence and extent of oxidation depends on the presence of oxygen, metals, enzymes, unsaturated lipids, prooxidants, antioxidants; exposure to light; type and physical state of the carotenoids present; severity and duration of processing; packaging material; storage conditions. Thus, retention of carotenoids has been the major concern in the preparation, processing and storage of foods. However, in recent years the effect of processing on bioavailability has been focalized. More than a century after their discovery, carotenoids continue to be intensely investigated in various areas. This article aims to give an overview of current knowledge in Food Science and Technology, which has bearing on the role of carotenoids in human health.

[Basic aspects and measurement of the antioxidant vitamins A and E]. / Aspectos básicos y determinación de las vitaminas antioxidantes E y A.

Vitamin E usually works as a biological antioxidant, preventing the oxidation of polyunsaturated fatty acids and proteins, for which it is considered an important protective factor in the development of diseases related to oxidative processes. Beyond its antioxidant properties, it has been involved also in genetic expression, mitochondrial metabolism, cell differentiation and immune system regulation. From the point of view of its antioxidant protection properties, values > or = 1200-1300 micrograms/dL are considered optimum levels (standardized according to plasmatic lipid levels). In relation to the beneficial advantage effects of vitamin E on primary or secondary atherosclerotic disease, data are not conclusive. Vitamin A is part of the organism's defense barrier against free radicals. Its antioxidant mechanism of action includes scavenging of single oxygen and thiol free radicals, and it also could be related to processes that involve genetic expression and cell differentiation. As an antioxidant, vitamin A plasmatic levels > or = 80 micrograms/dL are considered optimal. The highest risk of using this vitamin is related to its acute or chronic toxicity. Quantification of serum vitamin E (alpha tocopherol) and vitamin A (retinol) are made by high performance liquid chromatography (HPLC), method of high precision, sensitivity and reproducibility.

Curso Tópicos de Medicina Interna - aula 14 Molho de Tomate: boas notícias sobre um velho amigo / Course: topics in internal Medicine, 14th lesson: Tomato souce: good news about a old friend

O lycopene é um dos carotenóides mais ingeridos nas dietas dos países ocidentais, sendo o tomate (Solanum lycopersicum) cozido sua principal origem. Dentre os carotenóides, é o que tem maior capacidade quelante contra o singlet oxigêncio. Estudos experijentais e epidemiológicos sugerem efeitos protetores do lycopene em doenças cardiiovasculares e vários tipos de câncer, em especial no câncer de próstata

Bioavailability of carotenoids.

Our current knowledge about the bioavailability of provitamin A carotenoids in foods is insufficient, fragmentary and difficult to interpret. Past methods of estimating the vitamin A value of food carotenoids suffer both from uncertainty about the meaning of bioavailability and from the inadequacy of the indicators used in its determination. Reported conversion ratios of beta-carotene to vitamin A in humans in vivo, depending on conditions, range from 2:1 to 26:1 (microgram/microgram). Thus, the ratio of 6:1, devised by the World Health Organization, must be considered as a rough average estimate that is not applicable to all diets. Strategies to increase the dietary intake of carotenoid-containing foods should include measures to enhance carotenoid bioavailability.

[Investigation needs on carotenoids in Latin America]. / Necesidades de investigación en carotenoides en América Latina.

Many recent papers show the important role of bioactive phytochemicals to maintain a good health status. Among them the carotenoids are the best known. About 637 have been described and possibly 70 of them could have an important role in human health, 16 have been found in human brain in high amounts. Most of the studies have found relations between the carotenoids and chronic non-communicable diseases like several types of cancer, atherogenic disease and some degenerative pathology of the eye. This relation is mediated by genes and age. Studies of carotenoids are of scientific and economic interest for Latin America as many tropical products are high sources of these compounds. Therefore the first task is to analyze them and iniciate some evaluation on its metabolic availability. A coordinated regional work is proposed, in which 40 or 50 fruits and vegetables are analyzed in terms of the seven carotenoids most related to human health. At the same time it will be important to start epidemiological studies that will compare groups with different levels of consumption of fruits and vegetables and make chronic disease risk analysis. In some countries of the Latin American region, with the support of FAO and INFOODS, some courses and meetings are taking place so that in a short time period the carotenoid composition of the important regional foods will be completed and a carotenoid regional food composition table be published.

Biodisponibilidad de carotenoides / Carotenoid bioavability

La vitamina A y sus derivados conocidos como retinoides (de origen animal) y compuestos pro-vitamina A denominados carotenoides (de origen vegetal) son importantes en la prevención de cáncer, enfermedades crónicas y enfermedades relacionadas con la deficiencia de vitamina A; por tanto, es importante conocer la absorción, metabolismo transporte y almacenamiento de estos compuestos en humanos. Debido a lo complejo que ha sido la utilización de modelos humanos para estudiar la biodisponibilidad de carotenoides de fuentes naturales y sintéticas, recientemente se han desarrollado modelos aniomales que permiten avances significativos en áreas de poco conocimiento. Esta revisión pretende dar la mayor información acerca de la farmacocinética y el metabolismo de este nutriente que permita a los interesados utilizar el modelo más apropiado para los fines que persiga

Biodisponibilidade dos carotenóides do Buriti (Mauritia flexuosa L.) em ratos / Bioavailability of Carotenoids from Buriti (Mauritia flexuosa) L.) in rats

Considerando a magnitude da hipovitaminose A como problema de saúde pública no mundo e a disponibilidade de frutos ricos em pró-vitamina A na regiäo amazônica, determinou-se biodisponibilidade dos carotenóides do Buriti (Mauritia flexuosa L.) em ratos. Quarenta e oito ratos machos da linhagem Wistar (Rathus novergicus, var. albinus, Rodentia: Mamalia) recém-desmamados, com peso médio nicial de 33,8 + ou - 1,7g, foram distribuídos em cinco grupos, ou seja, Grupos: Deficintes, Controle 1200, controle 2400, Buriti 1200 e Buriti 2400. As Racöes foram elaboradas de acordo com a recomendaçäo do Committee on Laboratory animal Diets (1993). Após o período experimental de 28 dias, todos os animais foram sacrificados para a determinaçäo de vitamina A e caroteno no plasma e no plasma e no fígado. A menor concentraçäo de vitamina A hepática e plasmática foi observada no Grupo Deficiente. Por sua vez, as reservas hepáticas de vitamina A dos animais dos grupos Buriti 1200 e 2400 foram significativamente superiores quando comparados com os grupos Controle 1200 e 2400, respectivamente. Os resultados desse estudo demontraram ser o buriti uma fonte de pró-vitamina A altamente biodisponível, com eficiência relativa de 254,6 porcento (1200ER/Kg raçäo e 179,4 porcento (2400 ER/Kg raçäo) quando comparados com os respectivos grupos controle, indicando a maior biodisponibilidade dos carotenóides em doses próximas à recomendada.

Single- and multiple-dose-response relationships of beta-carotene in cystic fibrosis.

Concentrations of carotenoids are low in patients with cystic fibrosis (CF) and are associated with essential fatty acid deficiency and increased markers of inflammation. We conducted single- and multiple-dose studies of beta-carotene supplementation in patients with CF. Dose-proportional increases in beta-carotene concentrations were found, although clearance was independent of dose. Large doses of beta-carotene were necessary to achieve normal plasma levels.