Can You Raise Testosterone Levels Naturally And Without Medicine

Can You Raise Testosterone Levels Naturally And Without Medicine

Can a man's testosterone be boosted naturally?

Category: Health      Published: October 29, 2015

Testosterone molecule. Public Domain Image, source: Wikipedia.

Yes, but probably not in the way you are thinking, and only if the man has a healthy testosterone hormone system to begin with. Testosterone is an important hormone that plays many roles. In adult males, testosterone promotes muscle growth, physical energy, assertive and competitive moods, sperm development, libido, and emotional bonding. A man with abnormally low testosterone therefore experiences low energy, muscle weakening, mood problems, infertility, low libido, and emotional distance. In contrast, a man with a significantly higher-than-normal testosterone level can enjoy high energy, strong muscles, constructive moods, increased libido, and emotional closeness. For these reasons, boosting testosterone is generally desirable. However, if the testosterone level is too high, it can cause liver damage, uncontrollable moods, and cardiovascular disease. The ideal level of testosterone is therefore in the high end of the normal range. Fortunately, a man can only get dangerously high levels of testosterone if he dopes or if he has a rare disease. Testosterone doping, which involves internally taking in testosterone from an external source without medical necessity, is inherently dangerous and unhealthy because it raises testosterone levels too high. Testosterone doping is therefore illegal. As long as you don't resort to illegal drugs, any action that boosts your testosterone will elevate your levels while keeping you safely in the normal range, thereby allowing you to enjoy the benefits without the risks.

Let's look at various agents and actions that do and do not boost testosterone. Keep in mind that all of the concepts below assume that the man has a healthy testosterone hormone system, which includes a properly functioning hypothalamus, pituitary gland, and testicles. If a man has a non-functioning testosterone hormone pathway, no amount of effort will naturally boost his testosterone levels. Such a man has to undergo hormone replacement therapy in order to have normal health. Also note that testosterone is a hormone with a long-term action. This means that a higher testosterone level will only lead to higher energy, bigger muscles, and better moods if the testosterone levels remain consistently elevated for several weeks to months. Agents that may lead to a temporary elevation of testosterone followed by a dip have no effect on long-term testosterone levels and therefore do not give the desired long-term benefits.

Testosterone precursor pills do not boost long-term testosterone levels.
Testosterone precursor pills are commonly sold on the shelves of pharmacies and convenience stores, and are marketed as performance enhancing drugs, but the truth is that they don't work. Chemicals such as DHEA, androstenedione, and androstenediol are indeed used by your body to manufacture testosterone. But just because you have elevated amounts of androstenedione in your blood does not mean that your body will automatically convert it all into elevated amounts of testosterone. In fact, your body will convert almost none of the surplus precursor. If testosterone precursor pills actually worked like testosterone doping does, they would be pulled off the shelves and be made just as illegal as testosterone doping. A review article in the Journal of Athletic Training written by Michael E. Powers presents numerous studies which found that taking testosterone precursor pills does not increase long-term testosterone levels. Researchers found that excessive testosterone precursors in the male body are converted to estrogen and not testosterone. Therefore, taking DHEA pills, androstenedione pills, or androstenediol pills leads to elevated estrogen in males, not testosterone. Powers states,

As mentioned previously, ergogenic claims are based on the theory that precursor ingestion will result in increased testosterone levels, which would then stimulate an increase in muscle protein synthesis. However, at this time, there is no scientific support for this theory, as both DHEA and A'dione ingestion have failed to increase protein synthesis in groups of young men. Furthermore, 8 weeks of A'dione supplementation (300 mg/d) and resistance training failed to increase muscle-fiber cross-sectional area when compared with placebo ingestion and training.

Sexual activity does not boost long-term testosterone levels.
While sexual activity, whether visual or physical, does indeed raise testosterone levels for a few hours after the activity, it has no effect on long-term testosterone levels. Therefore sexual activity does not lead to long-term testosterone-induced health benefits such as increased muscle mass or heightened physical energy. In fact, a study by Helena C. Kraemer and her collaborators found that, "Mean testosterone levels were higher for sexually less active individuals."

Exercise does boost long-term testosterone levels.
Consistent exercise has been found to indeed raise long-term testosterone levels. This makes sense since two major roles of testosterone are to build up muscle tissue and to provide higher energy levels, both of which are used when exercising. High-intensity workouts have traditionally been thought to increase testosterone more than moderate-intensity workouts. However, research has found that although this may be true for the hour after the workout, there is no long-term difference in testosterone levels between a man pursuing high-intensity exercise and a man pursuing moderate-intensity exercise, all else being equal. For example, a study by Truls Raastad and his collaborators published in the European Journal of Applied Physiology states, "In conclusion, moderate- and high-intensity strength exercise did not differ with respect to prolonged (1-33 h) hormonal responses."

Adequate sleep does boost long-term testosterone levels.
Just like many other hormones, the release of testosterone follows a daily pattern of highs and lows that is regulated to some extent by the sleep cycle. Therefore, ongoing inadequate sleep, interrupted sleep, and irregular sleep all interfere with the release cycle and lead to lower testosterone levels. While developing good sleep habits will not boost testosterone much beyond the average healthy value, it will keep it from dipping below this value. A research letter by Rachel Leproult and Eve Van Cauter, published in the Journal of the American Medical Association, states, "Daytime testosterone levels were decreased by 10% to 15% in this small convenience sample of young healthy men who underwent 1 week of sleep restriction to 5 hours per night..."

Losing weight and eating a healthy diet does boost long-term testosterone levels.
In addition to a host of other health benefits, losing excess body weight and eating a nutritious, balanced diet indeed increases long-term testosterone levels. Eating too much sugar has been found to reduce testosterone levels, as has obesity in general. A review article by Giovanni Corona and his collaborators, published in the European Journal of Endocrinology, presents their compilation of the results of 24 research studies. The review article concludes that, "Overall, both a low-calorie diet and bariatric surgery are associated with a significant (P<0.0001) increase in plasma sex hormone-binding globulin-bound and -unbound testosterone levels (total testosterone (TT)), with bariatric surgery being more effective in comparison with the low-calorie diet...Androgen rise is greater in those patients who lose more weight..."

Reducing stress does boost long-term testosterone levels.
Ongoing psychological stress has been found to lower testosterone levels. Ongoing stress induces the body to release elevated amounts of the stress hormone cortisol, which signals to the body to make changes allowing it to better cope with the stress. One of these changes is the reduction of testosterone. The body makes this change so that energy and attention can be devoted to surviving the stress instead of building up body mass and enabling reproduction. Therefore, reducing the level of ongoing stress in a man's life can boost long-term testosterone levels. Note that over-exercising is a type of stress.

Taking vitamin D supplements and zinc supplements can boost long-term testosterone levels.
For those who aren't getting enough vitamin D or zinc, returning the amounts of these nutrients in the body to normal levels can indeed increase long-term testosterone levels. A study by Stefan Pilz and his collaborators, as published in the journal Hormone and Metabolic Research states, "In overweight men with deficient vitamin D status a significant increase in testosterone was observed after intake of 83 μg vitamin D daily for 1 year whereas there was no significant change in men receiving placebo."

Topics: boost testosterone, testosterone

Can You Raise Testosterone Levels Naturally And Without Medicine

Source: https://wtamu.edu/~cbaird/sq/2015/10/29/can-a-mans-testosterone-be-boosted-naturally/

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Lilyana Naturals Vitamin C

Lilyana Naturals Vitamin C

Generally, I loathe targeted advertisements—they feel like an instant invasion of privacy and give me the sneaking suspicion that computers are, in fact, taking over the world. But I'll give them a free pass for leading me to LilyAna Naturals, an all-natural skincare brand with a growing following on Amazon.

Presumably because I'm always on the hunt for best-selling skincare products on the e-commerce site, I was served an ad for LilyAna's best-selling eye cream. I was immediately struck by the number of five-star reviews the product had amassed. After a little digging, I realized that its clean, vegan, and cruelty-free products are Amazon best sellers with tens of thousands of glowing reviews.

Founded By: LilyAna Naturals CEO Retta Abraham says LilyAna's founder, who started the company in 2014, "prefers to remain anonymous."

Based In: Collinsville, MS

Pricing: $$

Best Known For: Being one of Amazon's top-reviewed skincare brands

Most Popular Product: Retinol Cream, Super C Serum

Fun Fact: "The brand was named after the two daughters of our director of operations, Lily and Anna Belle, both of whom grew up in Mississippi where the brand was founded," Abraham says.

Other Brands You'll Love: Burt's Bees, InstaNatural, Tree of Life

LilyAna, whose products are also sold through its own website, earned its rep on the strength of four rockstar products: a face cream, an eye cream, a retinol cream, and a vitamin C serum. In 2020, the super-hot brand was acquired by Abraham and Menna Samaha, a brother-sister investment team based in New York City. Despite the change in ownership, the brand hasn't strayed from its roots: LilyAna's products continue to be made in the United States and assembled in the teeny-tiny town of Collinsville, the brand's home since its inception.

Abraham says he and Samaha were drawn to LilyAna not just because of its "incredible amount of growth potential," but also because of the company's wholesome family vibe. "At its core, LilyAna Naturals is a homegrown family business," he says.

The secret ingredient of LilyAna's success? Its accessibility, Abraham says. "We want to provide skincare for all at an attainable price point," he explains; to whit, the brand's most expensive products are its $30 eye creams.

LilyAna Naturals' products are not only affordable, they're also safe and, seemingly, effective. LilyAna's mission, Abraham says, is to provide "supercharged formulas for immediate and lasting results"—with clean ingredients. In addition to being vegan and cruelty-free, LilyAna products are made without parabens, phthalates, sodium lauryl sulfate, sodium laureth sulfate, gluten, and artificial fragrances and colors.

If you've browsed any of the goods in LilyAna's Amazon shop—with their giddy reviews and low prices—you'd be forgiven for wondering if these products are the real deal, especially given the history of sketchy beauty buys for sale on Amazon. Although LilyAna's marketing isn't always clear about the concentration of active ingredients in its products, the experts we polled say this isn't a red flag.

"Many skin care brands do not share the concentrations of particular ingredients in their products to help keep their trade secrets," explains Joshua Zeichner, board-certified dermatologist and director of cosmetic and clinical research in dermatology at New York City's Mount Sinai Hospital.

This lack of information on ingredient potency can make it "difficult for the consumer to assess the product efficacy and how their skin might react to the the product," explain Gloria Lu and Victoria Fu, the cosmetic chemists better known by their moniker Chemist Confessions. But that hasn't stopped tens of thousands of Amazon reviewers from posting raves; in fact, Lu and Fu say they recommend LilyAna's vitamin C serums. (More on those in a sec.)

LilyAna began rapidly growing its product line in 2021, adding face washes; a toner, scrub, dark spot corrector and mask; and more vitamin C and retinol products that are racking up five-star reviews faster than Kylie Jenner changes hairstyles. So what are the brand's best products?

Read on for our favorite LilyAna Naturals products and reviews.

LilyAna Naturals Retinol Cream $23

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This retinol cream remains the hero product of the LilyAna Naturals brand, with tens of thousands of thumbs-up reviews on Amazon and glowing coverage in beauty editorial. The thick, luxurious texture of this hyaluronic acid–packed cream makes skin look plump and hydrated.

Although we're not sure how much retinol is in the product—and to that effect, how effective it is at treating wrinkles, fine lines, collagen loss, and uneven texture and tone—this is a decent nighttime moisturizer with lots of moisturizing botanical ingredients.

LilyAna Naturals Super C Serum $28

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If you're put off by that uncanny, expired-orange-juice scent of many vitamin C serums, you'll be delighted by this much more pleasantly scented—and affordable—serum, which gave our skin a noticeably brighter look within days.

At just $28, it contains "a solid amount of sodium ascorbyl phosphate, which is a good vitamin C alternative to the classic ascorbic acid serum," Lu and Fu say. "There's some nice data on SAP being a helpful active for oily, congested skin and pigmentation."

For best results, apply a thin layer of the serum, give it a few minutes to penetrate, and follow up with moisturizer or a hydrating sunscreen.

LilyAna Naturals Vitamin C Serum $23

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If you're a a relative skincare novice dipping your toes into the world of actives, LilyAna's original vitamin C serum is a great gateway product. With a lower concentration of vitamin C than the aforementioned Super C Serum, it's gentle on sensitive skin while providing a respectable antioxidant boost, improving skin tone and treating acne. We also appreciate the opaque packaging, which ensures the vitamin C doesn't degrade due to light exposure.

LilyAna Naturals Lavender Face Mask $23

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Although we haven't tried this hydrating brightening mask, we're eager to try it for a soothing self-care moment. Although it's said to be helpful for acne-prone and aging skin, the mask's mix of gentle plant extracts and oils seem best suited for hydrating balanced or dry skin and calming irritation.

LilyAna Naturals Vitamin C Eye Cream $30

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Antioxidant vitamin C is one of the few ingredients that can really, truly fade under-eye circles due to hyperpigmentation. This eye cream is formulated with a derivative of L-ascorbic acid to brighten under-eyes and stimulate collagen growth. It also has nourishing botanicals like licorice root extract (another hyperpigmentation fighter), aloe, jojoba oil, and rosehip seed oil.

If you're not used to using vitamin C under the eyes, keep in mind it can be a little drying with daily use. If you notice your under-eye area getting a little parched or flaky, try using it every other day, switching off with a more hydrating eye cream.

LilyAna Naturals Retinol Serum $28

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LilyAna's retinol serum is described as a more potent sister to its best-selling Retinol Cream. If you're a fan of the cream and want more bang for your buck, try this serum—or, better yet, layer the serum under the cream for maximum anti-aging effects.

Although the no. 1 best-selling status of LilyAna's Retinol Cream seems destined to endure, we think this serum is one of the better retinol offerings from the brand, including its lower-potency Retinol 0.5% Eye Cream and Retinol Face Wash.

LilyAna Naturals Super C Face Cream $26

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Love what vitamin C does for your skin but need a little more moisture than a serum can provide? Try this face cream with acne-fighting tea tree oil and salicylic acid.

Although it has a vaguely medicinal smell, the scent quickly dissipates after application, and the cream makes skin feel nourished and supple for hours. We dig.

LilyAna Naturals Eye Cream $30

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If potent, anti-aging eye creams have ever left you irritated and swearing off eye cream completely, this clean, no-frills eye cream might be for you. It's made mostly with good-for-skin, plant-based ingredients like aloe vera, coconut oil, rosehip seed oil, and vitamin E. It's simple and wholesome—which, if you don't have serious wrinkles or dark circles, may be just what you want from an eye cream.

LilyAna Naturals Face Cream $23

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One of the brand's four core products, the rosewater-scented Face Cream is a solid moisturizer for most skin types. It contains lots of lovely moisturizing ingredients—like carrot seed and meadowfoam seed oils and hyaluronic acid—plus antioxidant cranberry extract, pomegranate extract and vitamin C.

Although some Amazon reviewers complained that this cream didn't leave their dry skin feeling appropriately quenched, people with balanced, sensitive, or combination skin should get by just fine.

LilyAna Naturals Vitamin C Face Toner $23

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LilyAna's first toner smells like a delicious, citrus-y cocktail and leaves skin feeling refreshed and lightly hydrated, thanks to aloe vera, hyaluronic acid and niacinimide. Although the product comes in a (very generously sized) spray bottle, the nozzle didn't deliver the elegant, fine mist we were hoping it would; we found it easier to apply the toner with a cotton pad versus spritzing directly on our faces.

Lilyana Naturals Vitamin C

Source: https://www.byrdie.com/lilyana-naturals-amazon

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Is White Vitamin C Good For Pregnant Woman

Is White Vitamin C Good For Pregnant Woman

Vitamin C is needed to make collagen, one of the fibres that builds your baby's body. So, it's no surprise that your need increases during pregnancy. Fortunately, it's easy to get an adequate supply from a diet that includes plenty of fruit and vegetables.

Learn about the benefits of vitamin C in pregnancy, how it can help iron absorption and which foods are the best sources of vitamin C .

Why is vitamin C so important during pregnancy?

Not only does it boost your immune system and reduce your risk of suffering from iron-deficiency anaemia in pregnancy, Vitamin C is key to your baby's physical development too.

Vitamin C:

• Aids in the production of collagen, which supports normal growth, healthy tissue and wound healing¹
• Supports your baby's immune system²
• Helps your baby to absorb iron and build up stores for later use²

How much vitamin C do you need when you're pregnant?

The Reference Nutrient Intake (RNI) of vitamin C during pregnancy – the amount considered to be enough to meet most people's needs – is 50mg per day, which is 25% more than you would normally need³. A 100g portion of strawberries contain 57mg. If you decide to breastfeed your baby, you shouldn't need to make any dietary changes but it's a good idea to eat healthily. You can always talk to your midwife or healthcare professional if you'd like more advice. Read more about a healthy breastfeeding diet.

Since it is a water-soluble vitamin, it isn't stored by your body, which means a daily intake during pregnancy is essential. Fortunately, you can get all the vitamin C you need to support you and your baby by eating a healthy, balanced pregnancy diet that includes plenty of fruit and vegetables ⁴.

Which foods contain Vitamin C?

Foods rich in vitamin C include broccoli, cauliflower, kale, kiwi fruit, citrus fruits, red, green or yellow pepper, sweet potatoes, strawberries and tomatoes.

Peppers contain over twice as much vitamin C as oranges. Peppers contain around 126mg per 100g whereas oranges only contain 52mg of vitamin C per 100g ⁵ .

The graph below shows the amount of vitamin C you get from different foods⁶:

Food	Portion	Average nutrient quantity (mcg)
Red peppers	100g	126
Broccoli (best raw or steamed)	100g	79
Strawberries	100g	57
Cabbage (green or white raw)	100g	45
Citrus fruit (oranges are the best)	100g	42-52
Orange juice	100g	40
Tomatoes (best cooked)	100g	30
Spinach	100g	30

Steam and grill to boost your vitamin C intake

As with other water-soluble vitamins, the way you prepare and cook foods can affect the vitamin C content⁵.

Boiling can destroy some of the vitamin C within the foods you're preparing. To retain as much nutrient quality as possible, it's best to steam or grill your vegetables⁵. Or better still, eat them raw in salads.

Vitamin C boosts your iron absorption

Vitamin C helps the body absorb non-haem iron, the type found in plant sources such as spinach and chickpeas. Eating good sources of vitamin C with plant sources of iron during pregnancy can increase your daily intake considerably⁷. To get the most out of your diet, include fruit and iron sources within the same meal, whether it's adding chopped fruit to a salad or having a whole fruit for dessert⁷.

An adequate intake of iron is essential to support your increased blood volume and reduce the risk of iron-deficiency anaemia, and to help build up your baby's iron stores to support their learning and growth for the first 6 months of life². Read more about the importance of iron in your pregnancy diet.

Try increasing your intake with these vitamin C-rich snacks and meals:

• Porridge topped with sliced strawberries
• Sliced red peppers dipped in hummus
• Blackberry & raspberry ginger yoghurt pots
  
• Corn & courgette fritters with poached eggs
  
• Sesame-crusted tuna steaks with quinoa
  
• Sweet potato with homemade beans & feta
  
• Italian panzanella salad with roast chicken

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1. Maggini S et al. Essential Role of Vitamin C and Zinc in Child Immunity and Health. J Int Med Res 2010; 38: 386 - 414
2. NHS. Vitamins for children [Online] 2015 Available at: https://www.nhs.uk/Conditions/pregnancy-and-baby/Pages/vitamins-for-children.aspx [Accessed October 2017].
   
3. NHS UK. Iron-deficiency anaemia – complications [Online]. 2014. Available at: www.nhs.uk/Conditions/Anaemia-iron-deficiency-/Pages/Complications.aspx [Accessed June 2014]
4. Rumbold A, Crowther CA. Vitamin C supplementation in pregnancy. Cochrane Database Syst Rev 2, 2005.
5. Royal Surrey County Hospital (NHS). Check your iron intake pdf [Online]. 2012. Available at: www.royalsurrey.nhs.uk/Patients/Information-Leaflets/DownloadPDF?DocID=1233%2c1139%2c5%2c1%2cDocuments&MediaID=d863305d-d3d2-409b-abbb-841da008bcf2&Filename=PIN543_Check_your_iron_intake_w.pdf[Accessed June 2014]
6. Dietary reference values for food energy and nutrients for the United Kingdom. Committee on Medical Aspects of Food Policy. Report on Health and Social Subjects 41, 1991.
7. Department of Health. Nutrient analysis of fruit and vegetables [Online]. 2013. Available at: www.gov.uk/government/uploads/system/uploads/attachment_data/file/167942/Nutrient_analysis_of_fruit_and_vegetables_-_Summary_Report.pdf [Accessed June 2014]
8. Gov.UK. Nutrient Analysis of Fruit and Vegetables [Online]. 2013. Available at: www.gov.uk/government/publications/nutrient-analysis-of-fruit-and-vegetables [Accessed August 2014]
9. NHS UK. Vitamins and minerals [Online]. 2012. Available at: www.nhs.uk/Conditions/vitamins-minerals/Pages/vitamins-minerals.aspx [Accessed June 2014]

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Your baby's future health begins here

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Is White Vitamin C Good For Pregnant Woman

Source: https://www.aptaclub.co.uk/pregnancy/diet-and-nutrition/key-vitamins-and-nutrients/pregnancy-nutrients-vitamin-c.html

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How Much Vitamin C For Sinus Infection

How Much Vitamin C For Sinus Infection

BMC Complement Altern Med. 2013; 13: 110.

The effects of vitamins C and B12 on human nasal ciliary beat frequency

Jian Jiao

¹Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

²Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of Otolaryngology, Beijing, China

Na Meng

¹Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

²Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of Otolaryngology, Beijing, China

Hong Wang

¹Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

²Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of Otolaryngology, Beijing, China

Luo Zhang

¹Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, Beijing, China

²Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of Otolaryngology, Beijing, China

Received 2012 Oct 10; Accepted 2013 May 16.

This article has been cited by other articles in PMC.

Abstract

Background

This study was designed to investigate the effects of the vitamins C and B12 on the regulation of human nasal ciliary beat frequency (CBF).

Methods

Human nasal mucosa was removed endoscopically and nasal ciliated cell culture was established. Changes of CBF in response to different concentrations of vitamin C or vitamin B12 were quantified by using high-speed (240 frames per second) digital microscopy combined with a beat-by-beat CBF analysis.

Results

At the concentrations of 0.01% and 0.10%, vitamin C induced an initial increase, followed by a gradual decrease of CBF to the baseline level, while 1.00% vitamin C induced a reversible decrease of CBF. Vitamin B12, at the concentrations of 0.01% and 0.10%, did not influence CBF during the 20-min observation period, while a 1.00% vitamin B12 treatment caused a time-dependent but reversible decrease of CBF.

Conclusions

Treatment with vitamin C or vitamin B12 caused a concentration-dependent but reversible decrease of CBF in cultured human nasal epithelial cells. Therefore, it is necessary to choose a concentration that is safe, effective, and non-ciliotoxic when applying these drugs topically in the nasal cavity.

Keywords: Ciliary beat frequency, Vitamin C, Vitamin B12, Nasal epithelial cell

Background

Intranasal administration as a non-invasive route for drug delivery has generated much interest within the pharmaceutical industry in recent years. The nasal mucosa has many advantages as a potential site for both topical and systemic drug delivery, which include a large surface area for delivery, rapid onset of therapeutic effect, potential for central nervous system delivery, no first-pass metabolism, and, owing to its non-invasive nature, is likely to maximize patient comfort and compliance [1].

The safety of drug administration is an important parameter that must be taken into consideration during intranasal drug delivery. The nasal mucosa is lined by pseudostratified columnar ciliated epithelium. Coordinated beating of epithelial cell cilia with a normal pattern and frequency, which induces clearance of mucus from the airway, is the driving force of respiratory mucociliary transport. Impairment of the nasal mucociliary transport system can produce serious consequences because mucociliary transport is a major defense mechanism of the respiratory tract [2]. Therefore, investigation of the influence of nasally-administered drugs on ciliary activity is of great importance to assessing drug safety.

Vitamin C (ascorbic acid) is an important antioxidant in the respiratory tract, it is also an effective anti-inflammatory and anti-allergic agent; supplementation of vitamin C has been verified as an effective therapy for the treatment of certain respiratory diseases, including allergic rhinitis [3], chronic rhinosinusitis [4], and the common cold [5]. Since intranasal delivery route is commonly used for rhinologic drugs, we consider the intranasal route as a possible route for vitamin C supplementation, either alone or as an ingredient of certain drugs or medications.

Vitamin B12 (cobalamin) is necessary for the development of red blood cells, growth, and proper functioning of the nervous system [6]. Vitamin B12 deficiency is frequently found in elderly patients, with the main causes being pernicious anemia and food-cobalamin malabsorption. Management of vitamin B12 deficiency with cobalamin injections is currently well documented, but new routes for the administration of vitamin B12, both nasal and oral, are currently being developed [7].

The aim of the present study was to assess the ciliary toxicity of a range of concentrations of vitamin C or vitamin B12 treatment on cultured human nasal epithelial cells, so as to provide some advice for drug development and clinical medication.

Methods

Reagents

Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium/Ham's F12 (DMEM/F12), Hank's balanced salt solution (HBSS) and antibiotics were purchased from GIBCO BRL (Grand Island, NY, USA). Vitamin C was provided by Prof. Zinreich (Johns Hopkins Hospital, Baltimore, USA). Vitamin B12, human placental collagen, protease, and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Human nasal epithelial cell cultures

This study was performed at the Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital. The study protocol was reviewed and approved by the local ethics board of Beijing TongRen Hospital, Capital Medical University. Informed consent was obtained from all the participants.

Human uncinate process was endoscopically obtained from 8 adult patients who had cerebrospinal fluid rhinorrhea, nasopharyngeal angiofibroma or nasal septum deviation, without acute rhinosinusitis, chronic rhinosinusitis or allergic rhinitis.

Primary culture of human nasal epithelial cells was established using an adaptation of the methods published by Nlend et al.[8]. The dissected mucosa was incubated in 0.05% protease (type XIV) in DMEM overnight at 4°C. After protease treatment, epithelial cells were released by vigorous shaking and were harvested by centrifugation. The cells were plated on collagen-coated glass coverslips (human placental collagen, type IV) at a density of 5×10⁵/ml. The medium consisted of DMEM/F12 supplemented with 10 μg/ml insulin, 5 μg/ml transferrin, 0.36 μg/ml hydrocortisone, 20 ng/ml triiodothyronine, 7.5 μg/ml endochelial cell growth supplement, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were grown in an incubator at 37°C in 5% CO₂. The medium was changed every other day.

Measurements of ciliary beat frequency

Ciliary beat frequency was measured on the 1st, 3rd, 7th and 14th day of cell culture. Cell cultures between the 3rd to 14th days were used for CBF measurements in response to 3 different concentrations of vitamin C or vitamin B12 (0.01%, 0.10%, 1.00%), as well as HBSS. HBSS was used as a negative control to rule out the influence of the turbulence caused by changing the solution in the cell chamber.

The cultures were first allowed to equilibrate to 25°C for 10 minutes, and initially measured for three seconds to obtain a baseline CBF by rinsing with Locke Ringer's solution (136 mM NaCl, 5.6 mM KCl, 10 mM HEPES, 14.3 mM NaHCO₃, 1.2 mM MgCl₂, 2.2 mM CaCl₂ and 11.5 mM dextrose, pH 7.35). The cultures were then treated with the appropriate concentration of vitamin C, vitamin B12, or HBSS, and CBF was continuously recorded for three seconds every 1 minute for 20 minutes.

For high concentration of vitamins, namely, 1.00% vitamin C or 1.00% vitamin B12, after CBF measurements in vitamins solution for 20-min time-period, the cultures were followed by wash-out with the Locke Ringer's solution for another 20 minutes.

Measurements of CBF were made according to a previously published method [9,10]. Briefly, ciliated epithelial cells were viewed with an Olympus IX71 inverted microscope (Tokyo, Japan) equipped with a ×40, 1.3 NA, Ph 4, oil immersion objective. Imaging of cilia movement was carried out at 25°C and was achieved by directing the light forming the phase-contrast images to a high-speed CCD camera (TM-6710CL, Pulnix America, Sunnyvale, CA, USA). The camera was coupled with a frame grabber (Meteor, Matrox Electronic Systems, Dorval, Quebec, Canada) and recording software (StreamPix 3.16.5, Norpix Inc, Montreal, Quebec, Canada). The phase-contrast images of cilia movement were recorded at 240 frames per second. Images were evaluated using an image analysis program (IPLab v3.65a, Scanalytics, Inc., Fairfax, VA, USA).

The frequency of each ciliary beat cycle was calculated from the period of each cycle of the gray intensity waveform by a beat-by-beat analysis. The data analysis approach can measure the period [frequency (hertz) = 1/period] of each ciliary beat cycle to match the high temporal resolution of the image capture rate. The CBF was measured using a 3-second waveform (~720 frames) that was generated by the variation in gray level intensity of the phase-contrast images that resulted from the repetitive motion of the cilia. A region of interest, near the ciliary tip when the cilia were lying in the rest phase position, was selected and the average gray value was digitally extracted from the image data set and plotted with respect to time (i.e. frame number). The frequency of each ciliary beat cycle was determined from the period of each cycle of the gray intensity waveform.

Statistics

The data were expressed as means ± SEM. Statistical significance was assessed using a Repeated Measures ANOVA followed by Bonferroni's multiple comparison test. A value of p < 0.05 was considered statistically significant.

Results

Ciliary beat frequency on different culture day

On the 1st, 3rd, 7th, and 14th day of cell culture, CBFs of cultured cells were 7.32±1.55 Hz, 10.75±1.53 Hz, 10.73±2.15 Hz and 9.92±1.97 Hz, respectively. The CBF on the 1st day is significantly lower than the other days; in addition, the CBFs from the 3rd to 14th day are most close to the CBF of in vivo nasal epithelial cells, which is from 9 to 15 Hz. Therefore, we choose cell cultures from the 3rd to 14th day for CBF measurements in the following experiments.

Changes of CBF in response to vitamin C

The baseline CBF (CBF before treatment) for each of the three vitamin C groups and the HBSS group was 9.15 ± 1.07 Hz, 8.82 ± 1.29 Hz, 10.03 ± 1.67 Hz and 8.78 ± 1.56 Hz, respectively. There was no significant difference in baseline CBF within the groups (Figure1).

Changes of CBF in response to vitamin C. Ciliary beat frequency of cultured human nasal epithelial cells, measured before and every 1 min after treatment with 0.01% (n = 7, green dotted line), 0.10% (n = 7, blue dotted line) and 1.00% (n = 7, red dotted line) vitamin C, or HBSS (n = 7, black dotted line) over 20 minutes. *P < 0.05, vs. 0 min of each group.

Following treatment with 0.01% vitamin C, human nasal CBF increased over the first 2 min. The highest CBF, which was 10.13 ± 1.56 Hz, was observed at the first measurement point (1 min). CBF then gradually decreased to the baseline level. No distinguishable change in CBF relative to the baseline CBF was identified over the subsequent 18 min (Figure1).

Similarly, 0.10% vitamin C increased CBF over the first 4 min. The highest CBF, 10.07 ± 1.77 Hz, was again observed at 1 min after treatment. CBF then gradually decreased to the baseline level. No distinguishable change in CBF relative to the baseline CBF was identified over the subsequent 16 min (Figure1).

Ciliary beat frequency decreased significantly in response to the addition of 1.00% vitamin C. Initially, the CBF remained constant for approximately 10 min, and then continued to decrease over the next 10 min. At 20-min time-point after drug treatment, the CBF decreased to 6.84 ± 1.09 Hz (Figure1). However, after wash-out with Locke Ringer's solution, the CBF recovered rapidly in a time-dependent manner; CBF on 5-min time point during wash-out period recovered to 108.16 ± 3.32% of baseline CBF (Figure2).

Changes of CBF in response to vitamins wash-out. Ciliary beat frequency of cultured human nasal epithelial cells, measured before and after treatment with 1.00% vitamin C (n = 5), or 1.00% vitamin B12 (n = 5) over 20 minutes, followed by another 20 minutes wash-out with Locke Ringer's solution. *P < 0.05, vs. 20-min time-point of vitamin C treatment, #P < 0.05, vs. 20-min time-point of vitamin B12 treatment.

On treatment with HBSS, no discernible change in CBF relative to the baseline CBF was identified during the 20 minute measurement period (Figure1).

Changes of CBF in response to vitamin B12

Ciliary beat frequency changes in response to vitamin B12 are shown in Figures3 and ​ 2. Baseline CBF for the 0.01%, 0.10%, 1.00% vitamin B12 and HBSS groups was 8.96 ± 1.02 Hz, 9.35 ± 2.55 Hz, 10.23 ± 2.30 Hz and 9.07 ± 1.67 Hz respectively. No significant difference was found in baseline CBF within the groups (Figure3).

Changes of CBF in response to vitamin B12. Ciliary beat frequency of cultured human nasal epithelial cells, measured before and every 1 min after treatment with 0.01% (n = 8, green dotted line), 0.10% (n = 8, blue dotted line) and 1.00% (n = 8, red dotted line) vitamin B12, or HBSS (n = 8, black dotted line) over 20 minutes. *P < 0.05, vs. 0 min of each group.

Treatment with 0.01% and 0.10% vitamin B12 induced no significant change in CBF during the entire 20-minute measurement period. The CBF ranged from 8.96 ± 1.02 Hz and 9.35 ± 2.55 Hz (before treatment) to 9.46 ± 0.26 Hz and 8.95 ± 0.72 Hz (20 min after treatment) respectively (Figure3).

At a concentration of 1.00%, vitamin B12 induced a gradual decrease in CBF and statistical significance was identified at the last three 1-minute time points. At 20 min after 1.00% vitamin B12 treatment, CBF had decreased from 10.23 ± 2.30 Hz (baseline CBF) to 7.78 ± 2.34 Hz (Figure3).

At concentration of 1.00%, vitamin B12-induced CBF depression was also reversible, since the CBF was restored to the baseline level even after 1 minute of rinsing with Locke Ringer's solution, CBF at 1-min wash-out period reached to 97.68 ± 14.08% of baseline CBF (Figure2).

Following treatment with HBSS, no discernible change in CBF relative to baseline CBF was identified during the 20 minute measurement period (Figure3).

Discussion

Vitamin C is a well-known water-soluble and potent antioxidant, which exerts its antioxidant effect through scavenging reactive oxygen species by very rapid aqueous phase electron transfer, and preventing initiation of lipid peroxidation [11]. Vitamin C also has anti-inflammatory and anti-allergic effects; it might modulate the immune system by affecting the functions of phagocytes, proliferation of T lymphocytes, production of interferon, and gene expression of monocyte adhesion molecules [12]. In addition, vitamin C has been reported to be a biological regulator of the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP-dependent chloride channel that regulates respiratory tract epithelial surface fluid secretion, as well as controlling the hydration of mucosal surfaces and promoting effective mucociliary clearance. Topical administration of vitamin C to freshly-excised sinus and nasal mucosa was found to enhance chloride secretion, suggesting a potential for use as a therapeutic agent for the improvement of mucociliary clearance [13,14].

Given that oxidative damage, inflammation, immunological disturbance, and impairment of mucociliary clearance are common pathogenic factors associated with the pathogenesis of various rhinological diseases, such as chronic rhinosinusitis and allergic rhinitis, supplementation with vitamin C could have great potential as a novel therapy, either alone or in combination with traditional medications for the treatment of these diseases. In fact, some basic and clinical research has reported the effectiveness of vitamin C supplementation for the treatment of some respiratory diseases [3,4,15]. However, at present, most vitamin C delivery is by oral supplementation. Since most traditional medications for nasal disease, such as corticosteroid, antihistamines and vasoconstrictors, can be applied intranasally, we consider the intranasal delivery route as a possible route for vitamin C administration.

Vitamin B12 deficiency is common in the elderly (> 65 years of age), but is often unrecognized because of its subtle clinical manifestations, although they can be potentially serious, particularly from a neuropsychiatric and hematological perspective [16]. The main causes of vitamin B12 deficiency include pernicious anemia and food-cobalamin malabsorption, as well as poor diet, age-related gastric mucosal changes, and polypharmacy. Management of vitamin B12 deficiency with cobalamin injections is currently well-codified, but new routes of cobalamin administration (oral and nasal) are being studied, and several studies have confirmed the effectiveness of oral or nasal vitamin B12 supplementation serving as an alternative for intramuscular injection [17,18]. Compared with oral therapy, intranasal vitamin B12 supplementation could improve systemic absorption and ease of administration, avoid drug complexation with intrinsic factor in the process of oral absorption [18], and have a minimal potential for adverse systemic effects. However, although the intranasal route for vitamin B12 delivery has increasingly been used in clinical practice, the drug safety of topical form in vitamin B12 has been ignored.

Based on the common view that an effect on mucociliary clearance is one of the major areas of toxicity for nasal drug administration, the present study was designed to assess the ciliary toxicity of these two vitamins for intranasal application. CBF is a basic parameter for evaluation of ciliary activity, therefore, measurements of CBF changes in response to exogenous drugs on in vitro nasal epithelial cell cultures are commonly used to assess drug safety. However, the in vitro nasal epithelial cell culture do not represent in vivo nasal mucosa, it is reported that CBF of in vitro cultured cells may change over the culture time, moreover, the CBF between in vivo human nasal epithelial cells and cultured human nasal epithelial cells is different over the culture time [19]. Therefore, we first measured the CBF of cultured cells on different culture-time. Our results showed that CBF from the 3rd to 14th day is most close to the CBF of in vivo nasal epithelial cells, so we choose cell cultures from 3rd to 14th days for measurements of CBF in response to exogenous drugs.

We found that within concentrations from 0.01% to 0.10%, vitamin C had a stimulatory effect on human nasal CBF. However, at a higher concentration (1.00%), vitamin C produced an inhibitory but reversible effect on CBF. The effects of vitamin B12 on CBF were similar with that of vitamin C. At lower concentrations (from 0.01% to 0.10%), vitamin B12 has no significant effect on human nasal CBF. However, a higher concentration of vitamin B12, 1.00%, induced a time-dependent but also reversible decrease of human nasal CBF. These results raise the possibility of inducing loss of ciliary activity with higher concentrations of vitamins when applied intranasally, indicating the necessity of choosing a safe, non-ciliotoxic concentration when applying the vitamins topically in the nasal cavity.

The mechanisms of changes in CBF in response to vitamins treatment are still unclear. Airway CBF is mainly regulated by a variety of intracellular second messenger signaling mechanisms including cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and calcium [20]. cAMP activates an axonemal protein kinase A (PKA) to phosphorylation a dynein light chain to increase CBF; cGMP mediates a phosphorylation or dephosphorylation event to increase CBF via protein kinase G (PKG) and possibly PKA; calcium seems to increase CBF via an initial direct action on the axoneme and subsequently via cross talk to the cAMP or cGMP pathways. A significant amount of work is required to understand fully the mechanisms of regulation of CBF by these vitamins.

There are several other limitations of the current study. First, due to the short life-span of in vitro cultured epithelial cells, the effects of two vitamins on CBF were observed for only short period of 20 minutes; however, the use of vitamin C or B12 is not the single use but long term use in clinical medication, in addition, the present study showed a reversible decrease of CBF caused by single use of vitamins at high concentration, further indicating a possible long term ciliary toxicity in clinical use. Therefore, in the following studies, we are going to further assess the long term drug safety in vivo on laboratory animal models. Second, our experiments were performed on relatively normal nasal mucosa of patients, but not patients with acute or chronic rhinosinusitis. Since mucociliary transport and CBF make a difference in normal and inflammatory conditions, additional experiments for these inflammatory patients will provide more convincing results. In fact, we are in the process of performing further study on these patients so as to provide more detailed information for drug safety.

Conclusions

In summary, to the best of our knowledge, this study is the first to assess the influence of vitamin C and vitamin B12 on CBF in primary cultured human nasal epithelial cells. We have found that vitamin C, within a range of concentrations from 0.01% to 0.10%, has a stimulatory effect on human nasal CBF, while at a higher concentration (1.00%), it produces an inhibitory but reversible effect on CBF. Low concentrations (from 0.01% to 0.10%) of vitamin B12 have no obvious effect on CBF, while at a higher concentration (1.00%), it also induced a time-dependent but reversible decrease in human nasal CBF. These results do suggest the need for choosing a concentration that is effective, safe and non-ciliotoxic when applying these agents topically in the nasal cavity.

Abbreviations

CBF: Ciliary beat frequency; DMEM: Dulbecco's Modified Eagle's Medium; DMEM/F12: Dulbecco's Modified Eagle's Medium/Ham's F12; HBSS: Hank's balanced salt solution; CFTR: Cystic fibrosis transmembrane conductance regulator; cAMP: Cyclic adenosine monophosphate; cGMP: Cyclic guanosine monophosphate; PKA: Protein kinase A; PKG: Protein kinase G.

Competing interests

The authors declare that they have no competing interests.

Authors' contribution

JJ participated in the design of the study, carried out the ciliary beat frequency analysis, performed the statistical analysis and drafted the manuscript. NM and HW carried out the human nasal epithelial cell cultures and performed the ciliary beat frequency measurements. LZ participated in its design and coordination, conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the National Science Fund for Distinguished Young Scholars (81025007), National Natural Science Foundation of China (81100704 and 30973282), Beijing Natural Science Foundation (7131006), Beijing Nova Program (Z111107054511061), Specialized Research Fund for the Doctoral Program of Higher Education of China (20111107120004), The Capital Health Research and Development of Special (2011-1017-03), and Science Foundation for High-Level Medical Talents of Beijing Health System (2009-02-007).

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How Much Vitamin C For Sinus Infection

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663725/

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