TSK Koruyucu Hekimlik Bülteninde Ozon Tedavisi

Yazar: Özler M, Öter Ş, Korkmaz A. Ozon Gazının Tıbbi Amaçlı Kullanılması. TAF Prev Med Bull. 2009; 8(1): 69-74.
TAF Preventive Medicine Bulletin, 2009: 8(1)
Derleme/Review Article TAF Prev Med Bull 2009; 8(1):59-64

Ozon Gazının Tıbbi Amaçlı Kullanılması
[The Use of Ozone Gas for Medical Purposes]

ÖZET

Ozon (O3) üç oksijen atomundan oluşan renksiz, keskin kokulu doğal bir gazdır. Yer yüzeyi yakınlarında toksik ve kirletici olan ozon, stratosfer tabakasında zararlı ultraviyole ışınları süzücü rolüyle hayati önem taşır. Keşfinden sonraki ilk yıllarda dezenfeksiyon amacıyla kullanılırken yıllar içerisinde yapılan çalışmalar medikal kullanımını gündeme getirmiştir. Ozon tedavisi belirli bir miktarda ozon/oksijen karışımının vücut boşluklarına ya da dolaşım sistemine uygulanması olarak özetlenebilir. Ozon/oksijen gaz karışımı intravenöz, intramuskuler, intraartiküler, intraplevral, intrarektal ve intradiskal uygulanabildiği gibi topikal de uygulanabilir. En yaygın ozon uygulanma şekli majör otohemoterapidir. Bu yöntemde hastadan özel bir şişe içerisine alınan 50–270 ml kan ozon/oksijen karışımı ile belirli bir süre temas ettirilir ve daha sonra tekrar vücuda geri verilir (reinfüzyon). Ozon uygulaması esnasında oksidatif stres ve lipid oksidayonu sonucu oluşan hidrojen peroksit ikincil haberci gibi davranarak ozon tedavisinin biyolojik etkilerine aracılık eder. Tekrarlayan ozon uygulamaları sonucunda antioksidan sistem uyarılarak oksidatif strese karşı direnç gelişir. Ayrıca hücre membranında bulunan yağ asitlerinin oksidasyonuna bağlı olarak çeşitli sitokin düzeyleri de artar. Ozon tedavisi özellikle inflamatuar sürecin yoğun olarak yaşandığı ve immün sistemin ön planda yer aldığı fizyopatolojik durumlarda yardımcı tedavi yöntemi olarak kullanılmaktadır. Bu durumlardan bazıları yara iyileşmesi, yaşa bağlı makuler dejenerasyon, iskemik ve infeksiyöz hastalıklardır.

SUMMARY

Ozone (O3) is a colorless and sharp odorous natural gas that is composed of three oxygen atoms. Ozone, that is toxic and pollutant near earth’s surface, it is vital in stratosphere by absorbing harmful ultraviolet radiation. Although initial years after being discovered it was used for disinfection, studies conducted have come into question for medical usage of ozone. Ozone therapy may be summarized as administering a particular amount of ozone/oxygen mixture into body cavities or circulation. Ozone/oxygen gas mixture can be applied via intravenous, intramuscular, intraarticular, intrapleural, intrarectal and intradiscal as well as topically. Most frequent ozone administration is major autohemotherapy. In this method, 50-270 ml blood of patient is taken into a special bottle and after contacting with ozone/oxygen mixture for a particular duration, it is re-infused. During this period, hydrogen peroxide produced by oxidative stress and lipid oxidations mediates the biological effects of ozone therapy by acting as a second messenger. Repetition of ozone administration creates resistance against oxidative stress via inducing antioxidative system. Moreover, levels of several cytokine are increased depending on the fatty acid oxidation in cell membranes. Ozone therapy is used as an adjuvant therapeutic modality in the pathophysiological conditions where severe inflammatory processes and immune activation are involved. Some of the examples are wound healing, age-dependent macular degeneration, ischemic and infectious disorders.

Mehmet Özler, Şükrü Öter, Ahmet Korkmaz
GATA Fizyoloji AD
Anahtar Kelimeler: Ozon, ozon tedavisi, lipid peroksidasyonu.
Key words: Ozone, ozone therapy, lipid peroxidation.
Sorumlu yazar/ Corresponding author: Mehmet Özler.
GATA Fizyoloji AD Etlik, Ankara, Türkiye.
fizyomehmet@gmail.com
GİRİŞ
Ozon üç oksijen atomundan oluşan gaz halinde bir moleküldür. Oksijen molekülünün (O2) kararlı haline karşın, ozon (O3), kararsız bir moleküldür. Ozon gazını alman kimyacı Christian Friedrich Schönbein 1839 yılında keşfetmiştir. Ozon renksiz ve keskin kokulu bir gazdır. Keşfinden sonraki ilk yıllarda dezenfeksiyon amacıyla kullanılmıştır. 1860 yılında Monaco şehrinin su arıtma tesisinde dezenfeksiyon amacıyla ozon kullanılmaya başlanmıştır. Ozonun bu dezenfekte edici etkisi güçlü okside edici özelliğinden kaynaklanmaktadır. Sadece virüs ve bakterileri öldürmekle kalmaz tüm mikroorganizmalar ve toksinlerini de okside edebilir. Ozon ayrıca fenolleri, pestisitleri, deterjanları, kimyasal atıkları ve aromatik bileşikleri de etkili şekilde nötralize edebilir (1,2). Ozon kimyasal yapısı itibariyle radikal özelliği taşımamakla birlikte, florin ve persülfattan sonra, bilinen üçüncü en güçlü oksidan maddedir (3).

Ozon özellikle atmosferin üst tabakalarında oldukça bol bulunan bir moleküldür. Atmosferdeki ozonun %90’ına yakını, yer yüzeyinden yaklaşık 20–50 km yüksekte bulunan stratosfer tabakası içinde yer alır. Geri kalan %10’luk ozon miktarı ise 10–15 km’ler arasındaki troposfer tabakası içinde bulunmaktadır. Atmosferde stratosfer tabakası içerisinde bulunan ozon, ultraviyole radyasyonunun etkisiyle bir taraftan oluşurken, öbür taraftan da yok edilmektedir. Bu işlem ultraviyole radyasyonun değişik frekanslarında meydana gelir (4).

Ozon oluşumunu gösteren tepkime aşağıda gösterilmiştir.
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Çok reaktif bir gaz olan ozon canlılar için toksiktir. Akciğer ve gözler ozonun toksik etkisine en hassas organlardır. Gözdeki irritasyonu ve akciğere etkileri konsantrasyon, sıcaklık, nem ve maruz kalınan süreye bağlı olarak değişir. Düşük konsantrasyonda ozon inhalasyonu, boğazda irritasyon ve buna bağlı öksürüğe neden olabilir. Yüksek konsantrasyonlardaki inhalasyon ise bronşiyal mukoza ve pnömosit hücresi hasarına bağlı akciğer ödemine kadar varabilir (5,6).

Medikal Ozon ve Etki Mekanizması

Ozonun tıbbi amaçla kullanımının ilk olarak 1880 yılında Dr. John Harvey Kellogg (Battle Creek/Michigan/ABD) tarafından gerçekleştirildiğini yazan kaynaklar bulunmakla birlikte daha yaygın görüşe göre kabul edilen ilk tıbbi kullanımı Birinci Dünya Savaşı sırasında Alman askerlerinin kangren ve benzeri ciddi yaralanmalarını tedavi eden Dr. Albert Wolff’a dayanır. Bilimsel bir toplantıda ozonun tedavi edici bir ajan olarak gündeme alındığı ilk önemli organizasyon ise 1935 yılında Berlin’de toplanan 59uncu Alman Cerrahi Birliği (59th Meeting of the German Surgical Society) toplantısı olup, burada Dr. Erwin Payr “Cerrahi’de Ozon Uygulamaları” başlığı altında kendi vakalarından oluşan derleme türünde bir sunum yapmıştır (7,8). Bu tarihten sonra 80’li yıllara kadar, ozon tedavisini münferit olarak uygulayan çeşitli hekimler ve araştırmacılar bulunmaktadır. 1980’li yıllardan itibaren ise tıbbi amaçla ozon kullanımına yönelik gerek bilimsel çalışmalar, gerekse vaka serileri literatürde artmaya başlamıştır. Ozon tedavisi belirli bir miktarda oksijen/ozon karışımının vücut boşluklarına ya da dolaşım sistemine uygulanmasıdır; bu karışım intravenöz, intramuskuler, intraartiküler, intraplevral, intrarektal ve intradiskal uygulanabildiği gibi topikal de uygulanabilir (3). Ozon tedavisinin klasik uygulaması haline gelmiş olan yöntem 1974 yılında Wolff tarafından tarif edilmiştir. Bu yöntemde; bir miktar kan (50–270 ml) vücut dışına alınarak, ozona dayanıklı bir şişede 5-10 dakika oksijen/ozon karışımıyla temas ettikten sonra tekrar aynı kişiye geri verilir (ototransfüzyon) (3,9). Bu uygulama şekli majör otohemoterapi (HT) olarak adlandırılmaktadır. Bu tarihten günümüze, daha çok Avrupa’da olmak üzere milyonlarca ozon ototransfüzyon tedavisi yapılmıştır (10).

Ozon reaktif bir molekül olduğu için tıbbi amaçlı kullanımında dikkat edilmesi gereken bazı durumlar vardır:

Ozon, hiçbir zaman saf olarak verilmemeli ve belli oranda oksijenle karıştırılarak uygulanmalıdır. Bu karışımda oksijen %95’den az ozon %5’ten fazla olmamalıdır. Normal atmosfer havasının bu karışıma girmesi engellenmelidir. Çünkü ozonun reaktif özelliğinden dolayı hava ile teması sonucu toksik bir gaz olan nitrojen dioksit (N2O2) oluşabilmektedir. Ayrıca emboliye sebep olmaması için ozon gaz olarak damar sistemi içerisine verilmemelidir. Tüm işlemler sırasında ozona dayanaklı malzemenin (paslanmaz çelik, nötral cam ve teflon) kullanılması gerekmektedir (3).

Ozon, diğer gazlar (O2, CO2) gibi suda çözünebilir. Ozon oksijene göre 1,6 kat daha yoğun ve suda çözünürlüğü 10 kat daha fazla olan bir moleküldür. Saf suda diğer gazlar gibi Henry kanununa göre çözünür. Çözünmesi ısıya, basınca ve konsantrasyonuna bağlıdır. Biyolojik sıvılarda ise ozon oksijenden farklı olarak hızlıca biyomoleküller ile reaksiyona girer.

OzonPlazmaROTLOPTrombositLökositEritrositDiğer organlarKemik iliğiendotelBüyüme faktörlerinin salınmasıİmmünsistem uyarılmasıDokulara daha kolay Oksijen bırakma Antioksidan enzim miktarında artmaOksidatifstrese dirençli eritrosit yapımıve artmışkök hücre aktivasyonuNO salgılanmasında artma

Şekil 1. Ozon tedavisinin etkileri. (ROT: Reaktif oksijen türevleri, LOP: Lipit oksidasyon ürünü (Lipid oxidation products))
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Dolayısı ile HT esnasında uygulanan ozon/oksijen karışımındaki ozon afinite sırasıyla çoklu doymamış yağ asitleriyle, antioksidanlarla ve sistein gibi sülfhidril (SH) grubu taşıyan tiyol bileşikleri ile reaksiyona girer. Ozonun miktarına bağlı olarak karbonhidratlar, proteinler (dolayısıyla da enzimler), DNA ve RNA da bu reaksiyondan etkilenebilir. Tüm bu bileşikler ozon karşısında elektron donörü gibi davranarak oksitlenirler. Sonuçta süperoksit (O2-), hidrojen peroksit (H2O2) ve hipoklorik asit(HClO) gibi reaktif oksijen türevleri (ROT) oluşur. Bu reaksiyonlardan en önemlisi doymamış yağ asitlerinin oksidasyonudur. Ana reaksiyon aşağıdaki gibidir (3).

R-CH=CH-R’+O3+H2O→R-CH=O+R’-CH=+H2O2

Bu reaksiyonda her hidrojen peroksit ile birlikte iki de lipit oksidasyon ürünü (lipid oxidation products; LOP) oluşmaktadır (11,12). Lipit oksidasyon ürünleri için iyi bilenen örnekler şunlardır; lipoperoksil radikalleri, hidroperoksitler, malondialdehit, izoprostan, alkenaller ve 4-hidroksi-2,3-trans nonenal (HNE) (13-16).

Görülüyor ki, ozonun biyolojik etkilerinin ortaya çıkması için serbest radikallerin varlığı önemlidir. Serbest radikaller, çeşitli patolojik süreçlerin gerek başlatıcısı, gerek ara basamaklarda işe karışabilen, gerekse sonucunda ortaya çıkabilen reaktif maddelerdir. Bunlar, organizmada aerobik solunum sırasında mitokondride ve fagositlerde solunum patlaması gibi çeşitli fizyolojik durumlarda da oluşabilmektedir (17). Aerobik canlılar serbest radikallerin toksik etkilerinden korunmak için antioksidan sistemler geliştirmişlerdir. Non enzimatik olanlar; ürik asit, askorbik asit, protein (özellikle albumin), protein olmayan tiyoller, vitamin E ve biluribindir. Enzimatik olanlar ise süperoksit dismutaz (SOD), katalaz (CAT) ile glutatyon peroksidaz (GPx) glutatyon transferaz (GST), glutatyon (GSH) ve glutatyon redüktazdan (GR) oluşan glutatyon sistemidir (12). Son zamanlara kadar oksidatif stresin hücre hasarındaki rolü ve hastalıkların altında yatan patolojik süreçlere etkileri konusuna odaklanılmıştı. Patolojik süreçlerde oksidatif stresin artış mekanizmaları ve etkilerini açıklayan yüzlerce çalışma yapılmıştır (18-23). Yakın zamanda yapılan çalışmalarda ise oksidatif stresin bilinenin tersi etkilerinin de olabileceği görülmüştür. Bu çalışmalarda oksidasyon/redüksiyon (redox) reaksiyonlarının başta hücre içi haberleşme olmak üzere biyolojik mekanizmalarda rol aldığı gösterilmiştir. Artık açık olarak biliniyor ki gerek reaktif moleküller gerekse bunların çeşitli biyolojik moleküllerle reaksiyona girmesi sonucu ortaya çıkan oksidasyon ürünleri düşük konsantrasyonlarda (fizyolojik düzeylerde) hücrede önemli roller üstlenmektedir (23-26).

Ozonun biyolojik etkilerini açıklamak için yapılan çalışmalarda daha çok HT tedavisi model alınmıştır (27-30). HT esnasında uygulanan ozon/oksijen karışımındaki ozon plazmada hızla çözünür. Daha önce bahsedildiği gibi sıvılardaki çözünürlüğü fazla olan ozonun bir kısmı plazmada bulunan antioksidanlar ile reaksiyona girerek bunların miktarlarını azaltır. Bu anlık olaylar sırasında çeşitli ROT de oluşabilmektedir. Bu radikallerin yarı ömrü çok kısa olduğu için, daha kan hastaya geri verilemeden, yani ototransfüzyondan önce bunlar ortadan kalkarak yerlerini lipit oksidasyon ürünlerine bırakırlar. Bu ürünler, büyük oranda kandaki hakim hücre olan eritrositlerin membranlarının oksidasyonu ile ortaya çıkar. Eritrosit membranındaki doymamış yağ asitleri oksidasyona çok duyarlıdır. Yukarıda formülünü de gösterdiğimiz üzere, bu reaksiyonlar sırasında ortaya çıkan hidrojen peroksit, molekül yapısı itibariyle radikal olmayan oksitleyici bir moleküldür (3,12,31).

Hidrojen peroksitin ozonun tedavi edici etkinliklerinin en azından bir kısmından sorumlu - ikincil habercisi gibi davrandığı kabul edilmektedir. İlk etkilerinden biri eritrositlerde 2,3-difosfogliserat düzeyini artırma yoluyla hemoglobin-oksijen ayrışma eğrisinin sağa kaymasına ve böylece oksijenin dokulara daha kolay bırakılmasına neden olmasıdır. Plazmada konsantasyonu artan hidrojen peroksit kolayca hücrelerin içine diffüze olarak; lökosit ve endotelial hücrelerde çeşitli interferon, interlökin ve transforme edici büyüme faktörü (TGF) yapımını da artıran uyarıları tetikler (32). Lipit oksidasyon ürünlerinin yarı ömürleri ise saatlere varabilmekte, dolayısıyla ömrü çok kısa olan ROT’lerin ilk etkileri sonrasında ozonun gecikmiş etkilerinden sorumlu tutulmaktadır. Uzun yarı ömürlerinden dolayı bu ürünler ototransfüzyon ile vücuda verilmiş olur ve dolaşım yoluyla dokulara ulaşarak buralarda çeşitli biyolojik etkiler gösterirler (31,33,34).

HT tedavisi yapılmadan önce kanın antikoagülan verilerek hazırlanması gerekir. Çünkü ozon doza bağlı olarak trombosit fonksiyonlarının artışına neden olmaktadır. Trombosit fonksiyonlarındaki artışın bazı yararlı sonuçları da olmaktadır. Aktive olmuş trombositler içlerinde bulunan büyüme faktörlerini salarak iskemi ve ülserli hastalarda iyileşmeye olumlu katkı sağlar (12). HT sonrası ozonlanmış kanın vücuda verilmesi ile oluşan terapötik etkileri şekil 1’de gösterilmiştir (31).

Ozonun konsantrasyonuna bağlı olarak artan kuvvetli okside edici özelliği nedeniyle belli bir
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orandan sonra vücut için de toksik etkisi olabileceği gerçeğini unutmamak gerekmektedir. Doğal olarak, organizmadaki antioksidan savunma sistemleri ozon oksidasyonuna karşı koyacaktır. Plazmanın sahip olduğu geniş antioksidan kapasite ve eritrositlerdeki antioksidan enzimler nedeniyle, kan ozon toksisitesine karşı en dirençli dokudur. HT uygulamaları sırasında plazmada çözünen ozonun burada bulunan antioksidanlar (bilirubin, askorbik asit, SH grubu taşıyan glutatyon ve albumin) ile reaksiyona girerek bunların konsantrasyonunu azaltmaktadır (31). Öte yandan, HT sonucu ortaya çıkan ROT artışı ve antioksidanların azalması geçici bir durumdur. Bocci ve Carlo, yaptıkları çalışmada değişik dozlarda (20,40,60,80 μg/ml) ozon uygulanmış kanlarda dozla doğru orantılı olarak glutatyon ve total antioksidan seviyesinde azalma, lipit peroksidasyonu ve okside glutatyon düzeyinde artma olduğunu göstermiş, uygulamanın 20 dakika sonrasında ise antioksidan düzeylerinin eski haline döndüğünü tespit etmişlerdir (30).

Denilebilir ki, HT uygulaması sırasında tedavinin etkinliğini kanın toplam antioksidan gücü belirlemektedir. Kanın antioksidan kapasitesi düşük, ozonun konsantrasyonu fazla olursa şiddetli membran oksidasyonu sonucu eritrositler hemolize olur, tam tersi olduğunda ise ozondan beklenen ROS ve hidrojen peroksit yanıtı yeterli olmayabilir ve arzulanan terapötik etki görülemeyebilir. Ozon uygulamaları sonucunda oluşması beklenen ROT ve lipit oksidasyon ürünlerinin terapötik etki gösterebilmesi için belli bir konsantrasyonda olması gerekir (3). Bu açıdan, kanda bulunan antioksidanların önemi yapılan bir çalışma ile gösterilmiştir. Bu çalışmada eritrositler yıkanarak plazmadan uzaklaştırılmış ve değişik konsantrasyonlarda ozon uygulanmıştır. Yapılan değerlendirmelerde düşük 10-20 μg/ml ozon konsantrasyonlarındaki uygulamalarda bile eritrositlerin çoğunda hemoliz olduğu görülmüştür (28). Yapılan çalışmalarda ozonun terapötik konsantrasyonu 10-80 μg/ml olarak belirlenmiştir. Bu ozon konsantrasyonu Rice-Evans’ın tarif ettiği total antioksidan kapasiteyi %25’den fazla düşürmediği gibi azalan antioksidanlar 20 dakika sonra eski haline gelmektedir. (27,28,35,36).

Ozon uygulaması ile hem oksijenaz-1 (HO-1) enziminin de uyarıldığı bildirilmiştir. Bu enzimin artışından gerek ROT, gerekse yukarıda sözü edilen ılımlı eritrosit hemolizi sorumlu olabilir. HO-1, hem halkasının yıkım yolunda görev alan mikrozomal bir enzimdir ve yapımı oksidatif stres artışı, proinflamatuvar sitokinler ve nitrik oksit (NO) ile uyarılabilmektedir Bu enzim hem molekülünü biliverdin ve karbon monoksite (CO) parçalar. Son yıllarda HO-1 ile yapılmış birçok çalışmada bu enzimin; antioksidan antiapopitotik antiinflamatuar etkilerinin olduğu gösterilmiştir. Ozon uygulaması sonucu görülen en etkin HO-1 artışının aynı zamanda ozonun terapötik doz aralığı olarak da vurgulanan 20-80 μg/ml arasında ortaya çıktığı gösterilmiştir. Yine HO-1’in yanında ısı şok protein-70’in de arttığı gösterilmiştir (36-38).

Ozonun diğer bir uygulama şekli olan minör hemoterapide ise hastadan alınan 5 ml kan ile aynı miktarda 80-100 μl/ml konsantrasyonundaki oksijen/ozon karışımı bir dakika inkübe edilir. Bu süre zarfında ozonunun, yine aynı şekilde kanda önce çözünüp sonra da biyolojik moleküller ile reaksiyona girmesi beklenir. Sonrasında bu kan, gluteus kasına yavaşca enjekte edilir. Bu uygulama sonrasında kas içine enjekte edilen kanın doku derinliklerine ilerlerken pıhtılaşmasına rağmen hastalardan çok azı hafif şişme ve ağrıdan yakınmaktadır. Bu işlem esnasında anesteziye gerek yoktur. Tartışmalı olmakla birlikte, bu uygulamanın immünmodülatuar bir etkisinin olduğu iddia edilmekte ve etki mekanizması şu şekilde açıklanmaktadır: Enjeksiyon yerinde hafif derecede steril inflamasyon meydana gelmekte, bölgeye nötrofil ve monositler gelerek denatüre proteinleri ve parçalanmış eritrositleri fagosite etmektedir. Eğer kan içinde HCV, HBV ve HIV gibi virüsler var ise ozon tarafından inaktive edilip parçalanmış bu virüs atıkları bölgeye gelen bu immün hücreler tarafından ortadan kaldırılır. Böylece bu işlem bir çeşit aşı etkisi yaratır ve immün sistemi bu antijenlere karşı uyarır (3).

Medikal Ozon Tedavisinin klinik uygulamaları

Ozon tedavisinin özellikle inflamatuar sürecin yoğun olarak yaşandığı ve immün sistemin ön planda yer aldığı fizyopatolojik durumlarda tedavi edici etkisi şaşırtıcıdır. Ozon uygulamaları yara iyileşmesi, yaşa bağlı makuler dejenerasyon, iskemik ve infeksiyöz hastalıklarda yapılan vaka analiz çalışmalarında olumlu etkiler göstermiştir. Bunun yanında basit diş ve ağız enfeksiyonlarından hepatitlere kadar uzanan geniş bir aralıktaki çeşitli enfeksiyon hastalıklarında etkin olarak uygulanmaktadır (39-41). Martinez-Sanchez ve arkadaşları diyabetik ayak gelişmiş hastalarda yaptıkları çalışmada ozon tedavisinin etkinliğini değerlendirmişlerdir. Bu çalışmada ozon tedavisi uygulanan hastalarda antibiyotik tedavisi alanlara göre yara iyileşmesi hızlanmış, hastanede kalma süreleri kısalmış, kan şekeri düzeyleri daha iyi kontrol edilebilmiş ve antioksidan enzim düzeyleri artmış olarak bulunmuştur (42). Ayrıca çeşitli 62 www.korhek.org

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derecelerde artrit ve artroz vakaları ile romatizmal hastalıkları da kapsayan ortopedik hastalıklarda da faydalı etkiler rapor eden araştırmalar dikkat çekmektedir. Buna örnek olarak Mutu ve arkadaşlarının lomber disk hernisi olan hastalarda yaptığı çalışmayı gösterebiliriz. Bu çalışmada lomber disk hernisi olan hastalara oksijen/ozon karışımı disk içine enjeksiyonla uygulanmıştır ve gerek hasta memnuniyeti gerekse medikal olarak yapılan değerlendirmede bu tedavinin yararlı olduğu görülmüştür (43).

Medikal Ozon Tedavisinin Yan Etki ve Kontrendikasyonları

Ozon tedavisinin yan etkisi yok denecek kadar azdır. Şimdiye kadar bildirilen yan etkiler uygulama hatalarına bağlı lokal komplikasyonlardır. Bazı durumlarda ozon terapisi uygulanması sakıncalı olabilir. Bu durumlar: glukoz 6 fosfat dehidrogenaz enzim eksikliği (favizm), özellikle erken dönem olmak üzere hamilelik, anjiotensin çevirici enzim (ACE) inhibitörü tedavisi görenler, hipertiroidi, kanama bozukluğu, kontrol altına alınamayan kardiyovasküler hastalıklar ve ozona reaksiyon gösteren astım hastaları olarak sıralanabilir (44).

SONUÇ VE ÖNERİLER

Ozon tedavisinin tarihi süreci incelendiğinde ilginç bir gelişme gösterdiği ortaya çıkar. Bu molekül keşfedildikten bir müddet sonra sonra tıbbi amaçlı kullanılmaya başlanmıştır. Ozon tedavisinin ilkeleri bilimsel olarak belirlenmeden birçok klinik uygulama yapılmıştır. Yakın zamanda doz ve etki çalışmaları yapılmış uygulamalar daha bilimsel bir temele oturmuştur. Bununla birlikte halen ozon tedavisinin etki mekanizmasının birçok yönden açıklanmaya ihtiyacı vardır. Tüm dünyada devam eden deneysel ve klinik ozon tedavisi çalışmaları, yakın gelecekte mekanizmanın daha ayrıntılı açıklanmasına katkıda bulunacaktır. Ülkemizde pekçok uzmanlık alanından hekimin kullandığı ozon tedavisi ile ilgili deneysel çalışmalar özellikle hiperbarik oksijen tedavisi ile karşılaştırmalı olarak GATA Fizyoloji AD. Başkanlığı araştırma laboratuvarında devam etmektedir (45-48).

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48. Özler M, Ersöz N, Özerhan İH, Harlak A, Sadır S, Topal T, Öter Ş, Korkmaz A. Sıçanlarda oluşturulmuş peritoneal adezyon üzerine ozon tedavisinin etkisi. 2009; in press.
64 www.korhek.org

Yazının PDF Formatı http://www.korhek.org/khb/khb_008_01-69.pdf

Mevcut yazılı metin bilgilendirme amaçlıdır. Bilimsel verilerden elde edilmiş bilgilerdir. Konu hakkında uzman kişiler tarafından yönlendirilmeniz ve tedaviye yönelik işlemleri bir hekim kontrolünde uygulamanız veya uygulatmanız önerilir.

Kanserin Metabolik süreci

KANSERİN METABOLİK SÜRECİ



Boston'daki Beth Israel Hastanesi şef patoloğu Dr. Harold Dvorak, 'Gerçekte hiç kimse kanserden ölmez' diyor. Çoğunlukla organ iflası sonucunda hastalar kaybedilmektedir. Kanser hücresi normal hücrelerden 10 ila 15 kat daha hızlı çoğalır. Bunun sonucunda da fazla miktarda glukoza ihtiyaç duyar. Normal hücreler gibi glukozu yakmayan kanser hücresi glukozu fermente (oksijen kullanmadan yıkım) eder ve sonuç olarak ortama laktik asit salınımına ve aşırı metabolik yüke sebep olur. Laktik asit karaciğere giderek sitrik asit siklusu ile glukoza dönüşür ve karaciğerde glikojen olarak depo edilir.

Karaciğer ve tümör arasındaki bu kısır döngü sonucu oluşan metabolik yük ve enerji kaybı, karaciğerin aşırı çalışarak belirli bir zaman sonra kendisini tüketmeye başlaması ve bu durumun devam etmesi sonucu tükenmesi ile sonuçlanır.

Karaciğerin kanserde üstlendiği görev yukarıda da anlattığımız şekliyle hayati öneme haizdir ve önemi kesinlikle göz ardı edilmemelidir. Başarılı bir kanser tedavisi ancak sağlıklı ve fonksiyonlarını eksiksiz yerine getirebilen bir karaciğerle yapılabilir. Bu nedenledir ki karaciğerin üzerindeki yükü almamızı sağlayan, onun iş yükünü hafifleten ve bunun yanı sıra kanserde yine önemi kaçınılmaz olan amino asit içeriği ile genlerin tamirinde görev alan XP Tonics SLS’nin neden kullanılması gerektiğini açıkça, tartışmasız gözler
Mevcut yazılı metin bilgilendirme amaçlıdır. Bilimsel verilerden elde edilmiş bilgilerdir. Konu hakkında uzman kişiler tarafından yönlendirilmeniz ve tedaviye yönelik işlemleri bir hekim kontrolünde uygulamanız veya uygulatmanız önerilir.

KANSER METABOLİZMASI

KANSERİN METABOLİK SÜRECİ

Kanserden kimse ölmez, kanserin oluşturduğu ve çoğunlukla organ iflası sonucunda hastalar kaybedilmektedir. Kanser hücresi normal hücrelerden 10 ila 15 kat daha hızlı çoğalır. Bunun sonucunda da fazla miktarda glukoza ihtiyaç duyar. Normal hücreler gibi glukozu yakmayan kanser hücresi glukozu fermente (oksijen kullanmadan yıkım) eder ve sonuç olarak ortama laktik asit salınımına ve aşırı metabolik yüke sebep olur. Laktik asit karaciğere giderek CORI döngüsü ile glukoza dönüşür ve karaciğerde glikojen olarak depo edilir.

Karaciğer ve tümör arasındaki bu kısır döngü sonucu oluşan metabolik yük ve enerji kaybı, karaciğerin aşırı çalışarak belirli bir zaman sonra kendisini tüketmeye başlaması ve bu durumun devam etmesi sonucu tükenmesi ile sonuçlanır.

Karaciğerin kanserde üstlendiği görev yukarıda da anlattığımız şekliyle hayati öneme haizdir ve önemi kesinlikle göz ardı edilmemelidir. Başarılı bir kanser tedavisi ancak sağlıklı ve fonksiyonlarını eksiksiz yerine getirebilen bir karaciğerle yapılabilir. Bu nedenledir ki karaciğerin üzerindeki yükü almamızı sağlayan, onun iş yükünü hafifleten ve bunun yanı sıra kanserde yine önemi kaçınılmaz olan amino asit içeriği ile genlerin tamirinde görev alan XP Tonics SLS’nin neden kullanılması gerektiğini açıkça, tartışmasız gözler önüne sermektedir.

Hepatit C ve Ozone Therapy

Hepatitis C and Ozone Therapy
by Gérard V. Sunnen, M.D.

Abstract
Hepatitis C (HCV) is a global disease with an expanding incidence and prevalence base. Of massive public health importance, hepatitis C presents supremely challenging problems in view of its adaptability and its pathogenic capacity. The unique strategies that HCV utilizes to parasitize its host make it a formidable enemy and therapeutic interventions need considerable honing to counter its progress. Ozone, because of its special biological properties, has theoretical and practical attributes to make it a potent HCV inactivator.

History of the virus A form of hepatitis became recognized in the 1970's that resembled hepatitis B, serum hepatitis, and to a lesser extent hepatitis A, infectious hepatitis. It had, however, novel features, amongst them, a distinctive serological profile. In 1989, the genome of hepatitis C (HCV) was deciphered.

It is possible, by means of extrapolation from the genetic evolution of a virus, to approximate its age. Sequence genetic analysis points to the diversification of different HCV genotypes 200 to 400 years ago. Ancestors to these genotypes probably date back 100,000 or so years when viruses co-evolved with modern humans. Further analysis of genetic viral trees and Old and New World primates take the primordial forms of these viruses to primate speciation periods some 35 million years ago.

Today, in the context of human population growth, migration, and global travel, the hepatitis C virus has expanded its territories, geographically, and demographically. There is every indication that the evolution of this virus, in all its forms, is currently manifesting an accelerated phase.

Virion architecture and molecular biology The HCV particle is composed of a nucleocapsid containing its genome, an RNA single strand composed of approximately 9600 nucleotides, and its protein coating. The nucleocapsid is surrounded by an envelope which allows attachment and penetration into host cells. The genome encodes structural proteins designated as core (C), envelope 1 (E1), envelope 2 (E2), and P7 (unknown function), providing for virion architecture, and nonstructural proteins, mainly enzymes essential to the virion's life cycle, designated as NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Proteases release structural and nonstructural proteins. Helicases unwind viral nucleic acid. Polymerases replicate RNA. Within this genome is located a hypervariable region implying an area of intensive genetic fluidity and mutational potential. HCV displays great genotypic flexibility which makes for sophisticated evasiveness to host defenses.

The nucleocapsid is surrounded by an envelope, a lipid bilayer associated with a union of carbohydrates and proteins, glycoproteins. Up to 60% of the lipid component of the envelope is phospholipid and the remainder is mostly cholesterol. It possesses projections called peplomers which facilitate attachment to host cells. One protein on peplomers of the HCV particle which is thought to be instrumental in the attachment process is designated CD-81.

The sequence of nucleotides within the HCV genome shows significant variations. Strains obtained from different parts of the world, for example, may differ substantially in their structural and nonstructural protein compositions. This has lead to a system of classification of the HCV family into 6 genotypes (1 to 6), and approximately 100 subtypes (designated a, b, c, ect.). Genotypes vary from each other by a factor of 30% over the entire genome. Subtypes vary by about 20%. Genotypes 1 to 3 have global distribution, while genotype 4 and 5 are found mainly in Africa, and 6 is distributed in Asia. Importantly, genotype and subtype differences have shown varying susceptibility to antiviral therapy.

Within any one afflicted individual, HCV particles do not show a homogeneous population. Instead, they function as a pool of genetically variant strains known as quasispecies. This is due to the high replication error inherent in the function of the polymerase enzymes. Herein lies one of the important armaments of HCV. Continuously generated genetic diversity gives it great advantage in negotiating and conquering immune defense and therapeutic strategies. Furthermore, the antigenic differences between genotypes may have implications regarding the proper evaluation and the therapeutic regimen of patients.

Viral life cycle A freely circulating virion enters a host cell by binding to a cell surface receptor. In the case of HCV the host cell is a hepatocyte. However, bone marrow, kidney cells, macrophages, lymphocytes, and granulocytes may also be trespassed.

Once cell entry is achieved, the virion sheds its envelope to commence its replication. It binds to cellular ribosomes and released viral polymerase begins the RNA replication cycle. Newly formed nucleocapsids continue their assembly with the acquisition of new envelopes by means of budding through membranes of the cell's endoplamic reticulum. Newly formed virions may number in the range of 10 billion daily. The average life span of virions is in the order of a few hours.

Virions are then released into the general blood and lymphatic circulation, ready to infect new cells, re-infect already diseased cells, or a new host, mainly through bodily fluid transmission pathways. HCV RNA, as measured by polymerase chain reaction (PCR) may show 10 million or more virions per ml. As little as 0.0001 ml of blood may be sufficient to impart infection. The evolution of hepatitis C is characterized by phases of accentuated viremia punctuated by periods of relative quiescence. The presence and timely detection of these viremic waves may offer novel therapeutic considerations.

Clinical and laboratory manifestations Hepatitis, from anyone of the several viruses capable of inducing liver inflammation, produce a spectrum of clinical and laboratory manifestations. Hepatitis C distinguishes itself by the low incidence of acute phases and by the high incidence of progression to chronicity. Acute hepatitis C progresses from exposure, to incubation, to pre-icteric, icteric, and convalescent phases. With an incubation period of about 6 weeks, the first and sometimes only symptoms include weakness, fatigue, indolence, headache, nausea, poor appetite, and vague abdominal pain. The pre-icteric period extends from the onset of symptoms to the appearance of jaundice, ranging usually from 2 to 12 days. The icteric phase corresponds to the declaration of jaundice and darkened urine. The convalescent phase is marked by the gradual disappearance of symptoms.

Chronic hepatitis C is characterized by the presence of HCV RNA and the elevation of liver enzymes for 6 months or longer. Patients may be asymptomatic, or at times suffer an acute exacerbation with a return of symptoms. Approximately 75% of acutely ill patients continue into a chronic phase evidenced by parameters of viral presence.

Hepatitis C can only be distinguished from other viral hepatic conditions by serological and virological determinations. Liver enzymes characteristically affected by HCV infection include serum alanine transfesferase (ALT), aspartate aminotransferase (AST), gamma- glutamyl transpeptidase (GGTP), and alkaline phosphatase; in addition, there may be abnormalities in bilirubin, serum albumin, prothrombin time, and platelet density.

Cirrhosis, a diffuse disruption of liver tissue architecture with regenerative nodules surrounded by fibrosis, is an important sequel to hepatitis C. Within 20 years post HCV infection 20 to 25% of patients will develop cirrhosis. Hepatic decompensation ensues with ascites as the salient marker.

Hepatocellular carcinoma, another notable outcome of HCV infection is present in approximately 5% of patients post infection. The presence of cirrhosis is central to its genesis. Although the mechanisms by which cirrhosis ushers carcinoma are unknown, it is likely that chronic inflammation and the sustained pressure of cellular regeneration play important roles.

Up to 10% of patients appear to have fully conquered the disease. HCV antibodies are undetectable, as is HCV RNA. Liver enzymes are fully normalized, but liver biopsy may show lingering areas of stagnant inflammation and spotty necrosis. It is thus possible for host immunocompetence to vanquish HCV infection and therapeutic strategies aim to assist the host immune system to achieve this goal.

Immunological response to the virus HCV particles are detected early in the infection, usually 1 to 2 weeks following exposure. Antibodies to HCV core, nonstructural, and envelope elements appear about 6 weeks after exposure. A broad range of cytokines are mobilized. Cellular immunity is activated with broad recruitment of neutrophils, natural killer (NK), macrophages, and CD4 and CD8 T helper cells.

Current and experimental treatment strategies As of this date the main treatment strategies for hepatitis C include interferon and ribavirin. Interferons are natural cellular products which activate macrophages, neutrophils and natural killer cells. There is controversy as to interferon's biological effects, be they mostly immunoregulatory or directly antiviral. Ribavirin is a guanosine analog that represses messenger RNA formation thus inhibiting the replication of many DNA and RNA viruses. It is, however, mutagenic to mammalian cells. Ribavirin and interferon have significant medical and psychiatric side effects.

Treatment response is defined as undetectable viral load 6 months following therapy. Contemporary detection methods of quantitative HCV RNA determinations are capable of detecting approximately 1000 viral copies per serum ml.

Resistance to antiviral therapies is a particularly vexing problem in anti HCV treatment. Novel and experimental antiviral compounds include inhibitors of protease, polymerase and helicase.

Vaccine development needs to take into account HCV's antigenic rainbow and its high mutability. High mutation rates in this condition implies a dauntingly diverse and variable array of viral antigenic components. It is estimated, for example, that HCV mutates significantly in its own host approximately a thousand times a year. This implies that within any one afflicted individual there exists an awesomely large array of viral quasispecies, which in turn creates commensurate difficulties in the creation of effective vaccines.

Ozone: Physical and physiological properties Ozone (O3) is a naturally occurring configuration of three oxygen atoms. With a molecular weight of 48, the ozone molecule contains a large excess of energy. It has a bond angle of 127° and resonates among several forms. At room temperature, ozone has a half life of about one hour, reverting to oxygen. A powerful oxidant, ozone has unique biological properties which are being investigated for applications in various medical fields. Basic research on ozone's biological dynamics have centered upon its effects on blood cellular elements (erythrocytes, leucocytes, and platelets), and to its serum components (proteins, lipoproteins, lipids, carbohydrates, electrolytes). Administrating increaing dosages of ozone to whole blood shows that beyond a certain threshold there is a rise in the rate of hemolysis. This threshold, depending upon various parameters, begins to be reached at 40 to 60 micrograms per milliliter, and becomes significant when higher levels are attained. Precise ozone dosing capacity is therefore essential in clinical practice and research.

Leucocytes show good resistance to ozone because they have enzymes which protect them from oxidative stress. These enzymes include superoxide dismutase, glutathione, and catalase. Research has shown that platelets also maintain their integrity after ozone administration. In ozone therapy, the doses applied to blood are gauged to avoid disruption of its cellular elements. Serum components remain viable during ozone therapy. Lipid and protein peroxides, produced in small amounts by ozonation, have demonstrable antiviral properties. Interestingly, ozone tends to stimulate leucocyte function and cytokine production. Ozone increases the oxygen saturation (p02) in erythrocytes and enhances their pliability so that capillary circulation is facilitated.

Ozone: Antiviral properties Recently, there has surged renewed interest in the potential of ozone for viral inactivation. It has long been established that ozone neutralizes bacteria, viruses, and fungi in aqueous media. This has prompted the creation of water purification processing plants in many major municipalities worldwide.

Ozone's antiviral properties may also be applied to the treatment of biological fluids, albeit in technologically and physiologically appropriate ways. Indeed, it is noted that ozone, administered in such dosages designed to respect the integrity of blood's cellular and constituent elements, is capable of inactivating a spectrum of viral families.

Some viruses are much more susceptible to ozone's action than others. It has been found that lipid-enveloped viruses are the most sensitive. This group includes, amongst others, HCV, Herpes 1 and 2, Cytomegalus, HIV1 and 2.

The envelopes of viruses provide for intricate cell attachment, penetration, and cell exit strategies. Peplomers, finely tuned to adjust to changing receptors on a variety of host cells, constantly elaborate new glycoproteins under the direction of E1 and E2 portions of the HCV genome. Envelopes are fragile. They can be disrupted by ozone and its by-products.

In HCV, viral load appears to be a major factor in the invasiveness and virulence of the disease process. Preliminary research has shown that reduction of viral load in Hepatitis C by means of ozone therapy can significantly normalize hepatic enzymes and improve measures of global patient health. Volunteers administered ozone therapy according to the method outlined below achieved a viral load reduction in the order of 5 log, or 99.9%, along with a normalization of liver enzyme levels.

Ozone: Clinical methodology Ozone may be utilized for the therapy of a spectrum of clinical conditions. Routes of administration are varied and include external and internal (blood interfacing) methods. In the technique of ozone major autohemotherapy for hepatitis C, an aliquot of blood is withdrawn from a virally-afflicted patient, anticoagulated, interfaced with an ozone/oxygen mixture, then re-infused. This process is repeated serially until viral load reduction is documented.

The aliquots of blood range from 50 ml. to 300 ml. Ozone dosages and treatment frequency vary according to treatment protocols. The reason aliquots of blood are treated and not, as one would propose, the entire blood volume, is that in the latter case the total ozone dosage administered would exceed toxic limits.

The average adult has 4 to 6 liters of blood, accounting for about 7% of body weight. How can the viral load reduction observed via ozone therapy be explained in the face of a technique that treats relatively small amount of blood, albeit serially?

Ozone: Possible mechanisms of anti-viral action
The viral culling effects of ozone in infected blood may recruit the following mechanisms:

Denaturation of virions through direct contact with ozone. Ozone, via this mechanism, disrupts viral envelope proteins, lipoproteins, lipids, and glycoproteins. The presence of numerous double bonds in these unsaturated molecules makes them vulnerable to the oxidizing effects of ozone which readily donates its oxygen atom and accepts electrons in these redox reactions. Double bonds are thus reconfigured, molecular architecture is disrupted and widespread breakage of the envelope ensues. Deprived of an envelope, virions cannot sustain nor replicate themselves.

Ozone proper, and the peroxide compounds it creates, may directly alter structures on the viral envelope which are necessary for attachment to host cells. Peplomers, the viral glycoproteins protuberances which connect to host cell receptors are likely sites of ozone action. Alteration in peplomer integrity impairs attachment to host cellular membranes foiling viral attachment and penetration.

Introduction of ozone into the serum portion of whole blood induces the formation of lipid and protein peroxides. While these peroxides are not toxic to the host in quantities produced by ozone therapy, they nevertheless possess oxidizing properties of their own which persist in the bloodstream for several hours. Peroxides created by ozone administration show long-term antiviral effects which serve to further reduce viral load. This factor may explain in part the reason for the fact that ozonated blood in the amount processed in usual treatment protocols is able to reduce viral load values in the total blood volume.

Immunological effects of ozone have been documented. Cytokines are proteins manufactured by several different types of cells which regulate the functions of other cells. Mostly released by leucocytes, they are important in mobilizing the immune response. It has been found that ozone induces the release of cytokines which in turn activate a spectrum of immune cells. This is likely to constitute a significant avenue for the reduction of circulating virions.

Ozone action on viral particles in infected blood yield several possible outcomes. One outcome is the modification of virions so that they remain structurally grossly intact yet sufficiently dysfunctional as to be nonpathogenic. This attenuation of viral particle functionality through slight modifications of the viral envelope, and possibly the viral genome itself, modifies pathogenicity and allows the host to increase the sophistication of its immune response. The creation of dysfunctional viruses by ozone offers unique therapeutic possibilities. In view of the fact that so many mutational variants exist in any one afflicted individual, the creation of an antigenic spectrum of crippled virions could provide for a unique host-specific stimulation of the immune system, thus designing what may be called a host-specific autovaccine.

Summary
Viruses are far from being static entities. As quintessential intracellular parasites they have developed, through millions of years of cohabitation with their hosts, astoundingly sophisticated structures, survival, and propagation mechanisms. They have adapted, modified their biological strategies, and evolved impressive genetic diversity and mutational capacity to cope with the changing ecology of planetary life.

HCV has an extremely high rate of mutation and within any one individual there may exist millions of antigenic quasispecies. The disease process is marked by periods of viral quiescence alternating with viremic waves whereby billions of virions are poured into the blood and lymphatic reservoirs. Their astounding numbers stress the immune system relentlessly and produce an inexorable compromise in all parameters of its functioning.

Viral load reduction by means of ozone blood treatment alleviates immune system fatigue. Ozone-mediated viral culling may be achieved by anyone of a number of possible mechanisms. Direct virion denaturation, peplomer alteration, lipid and protein peroxide formation, cytokine induction, host pan-humoral activation, and host-specific autovaccine creation are suggested mechanisms. Due to the excess energy contained within the ozone molecule, it is theoretically likely that ozone, unlike antiviral options available today, will show effectiveness across the entire genotype and subtype spectrum.

Ozone embodies unique physico-chemical and biological properties which suggest an important role in the therapy of hepatitis C, either as a monotherapy, or as an adjunct to standard treatment regimens.

BIBLIOGRAPHY

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KANSERLE YAŞAM

      Zaman hızla geçip gidiyor ve kanser hastalığı yol almaya devam ediyor. Kanserojen maddelerdeki artış, genetik hasar konusundaki başarımız insanlığın en büyük ve en kötü illetini besleyip duruyor. Dr.Levent KARAFAKI
      Bize kalan ise batıda geliştirilecek tedavileri bekleyerek umudumuzu korumaya çalışmak. Bu arada unuttuğumuz en önemli kavram ise yaşamak. Nasıl olmalı da kanserle yaşamayı öğrenmeliyiz. Hastalarımın büyük kısmında gördüğüm etki bıkkınlık, bitkinlik ve en sonunda ne olacaksa bir an önce olsun da kurtulayım psikolojisi.Dr.Levent KARAFAKI
      Bu dünya hiçbirimize kalmayacak. Ama yaşamı zenginleştirmek ve haz almak bizim elimizde. Özellikle kanser hastalarında ki aslında tüm hastalarda en önemli olan yaşam sevgisi. Bu olmadan gribi bile atlatabilme gücüne sahip değiliz. Atatürk'ün bir sözü geliyor aklıma muhcat olduğunuz kudret damarlarınızdaki asil kanda mevcuttur. Asıl anlamı farklı olsa da sağlık için de bu cümlenin çok önemli olduğunu düşünüyorum. Yani hastalıkları iyileştirme gücü aslında kanımızda yani vücudumuzda mevcut. Sadece bizler bu gücü nasıl ortaya çıkarabileceğimizi bilmiyoruz. Asıl tedavi yolu buradan geçtiği halde biz bütün yükü ilaçların üzerinde bırakarak kendi bağışıklık sistemimiz ile iyileştirebilme gücümüzü ortaya çıkarmıyoruz.
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Şimdilik esen kalın yazının devamı nisanın son haftasında devam edecek.......

Dr.Levent KARAFAKI

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C VİTAMİNİNİN T HÜCRELER ÜZERİNE İMMÜNOMODÜLATÖR ETKİSİ

Immunology Letters 98 (2005) 63–72
Mega-dose Vitamin C modulates T cell functions in Balb/c mice only
when administered during T cell activation
Kahwa Noha,b, Hyunja Lima,b, Sung-kyu Moona,b, Jae Seung Kanga,b, Wang Jae Leea,b,
Dongsup Leeb, Young-il Hwanga,b,∗
a Tumor Immunity Medical Research Center, Seoul National University College of Medicine, Seoul 110-799, South Korea
b Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu,
Seoul 110-799, South Korea
Received 5 August 2004; received in revised form 20 October 2004; accepted 21 October 2004
Abstract
Previously we reported that a mega-dose of Vitamin C enhanced the initial stage of delayed-type hypersensitivity reaction in Balb/c mice.
In this study its effects were further evaluated as follows. Mice were administered Vitamin C intraperitoneally at 0.625 mg/day or at 5 mg/day
for variable days before, during, or after being sensitized with DNFB. T cells were isolated in each group and examined. When stimulated
antigen-specifically or non-specifically in vitro, mice showed elevated thymidine uptake and a shift of cytokine secretion profiles toward Th1,
i.e., elevated levels IL-2, TNF-
, and IFN-, and lowered level of the Th2 cytokine IL-4, only when Vitamin C was administered during
sensitization. T cells from those mice administered Vitamin C before sensitization or after challenge showed the same T cell properties as those
from PBS-treated mice. Mice were also given 0.625 mg/day of Vitamin C during primary and/or secondary immunizations with KLH and
secondary specific antibody titers in sera were measured. The total specific antibody titer was lowered in Vitamin C-treated animals whenever
treatments were administered, and this was entirely attributed to decreased levels of IgG1 and IgE antibodies. Based on these results, we
suggest that an exogenously administered mega-dose of Vitamin C shifts immunity in Balb/c mouse toward Th1 and that these affects occur
only when Vitamin C is administered during T cell activation.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Mega-dose; Vitamin C; Th1 polarization; Delayed type hypersensitivity; T cell activation; Isotype
1. Introduction
Vitamin C acts as an electron donor for many kinds of human
enzymes, facilitates iron transport, and is regarded as one
of the most important physiological antioxidants [1,2].In addition,
VitaminCexerts several diverse effects on the immune
system. It increases neutrophil motility [3,4] and phagocytic
function [5] in human. Macrophage functions in mice such as
chemotaxis, phagocytosis, and superoxide anion production
are enhanced by several antioxidants including Vitamin C
[6]. Increased proliferation of T cells [7,8], and inhibition of
∗ Corresponding author. Tel.: +82 2 740 8209; fax: +82 2 745 9528.
E-mail address: hyi830@snu.ac.kr (Y.-i. Hwang).
various forms of T cell death [9] and Fas-induced apoptosis
of monocytes [10] by Vitamin C have also been reported. The
increased cytotoxic activity of natural killer cells in humans
is another example of an effect ofVitamin C supplementation
[11,12].This biologically important micronutrient should be
exogenously administered in diets or as tablets to those, including
human, that lack terminal enzyme l-gulonolactone
oxidase in the Vitamin C synthetic pathway [13], and thus
cannot make Vitamin C themselves.
The recent recommended daily allowance (RDA) for
ascorbic acid by National Academy of Sciences is 75 mg and
90 mg per day for women and men, respectively. Nevertheless,
several authors have claimed that this dose is inadequate
and have suggested that the RDA be increased. For example,
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.imlet.2004.10.012
64 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Levine et al. [14] suggested 200 mg/day to maintain optimal
blood concentrations. Considering that low concentrations of
serum ascorbate are related to increased mortality [15,16], the
recent RDA could be below that actually needed to maintain
health.
Meanwhile, despite many controversies about its beneficial
effects, the usage of even higher doses of Vitamin C at
the level of grams per day has been suggested for both preventative
and therapeutic purposes in a number of diseases,
including cancer [17], atherosclerosis [18], viral infections
[19], H. pylori infection [20], and acute pancreatitis [21]. In
addition, supplement users regularly consume more than 1 g
of Vitamin C per day [22]. However, the effects and mechanisms
of such ‘mega-doses’ of Vitamin C have not been well
defined.
Previously we reported that exogenously given megadose
of Vitamin C augment the initial stage of delayedtype
hypersensitivity (DTH) response against 2,4-dinitro-1-
fluorobenzene (DNFB) in Balb/c mice [23]. In this study, we
further analyzed the ways in which mega-dose Vitamin C
modifies the functions of T cells in mice. When mega-dose
Vitamin C was administered during sensitization, activated
and memory T cells were rendered more vulnerable to both
ex vivo antigen-specific and non-specific stimuli and their
cytokine secretion profiles were shifted to Th1. Furthermore,
vitamin C also affected humoral immune response against
keyhole limpet hemagglutinin (KLH), by critically lowering
specific serum IgG1 and IgE levels.
2. Materials and methods
2.1. Mice
Seven-week-old male Balb/c mice were purchased from
BioLink (Seoul, Korea) and housed in animal facilities at
Seoul National University, College of Medicine (Seoul, Korea).
They were kept under a 12 h light–dark cycle and all
the animals were given mice chow and tap water ad libitum
through the experimental period.
Experimental groups were administered Vitamin C
(Sigma, St. Louis, MO) intraperritoneally at a daily dose
of 0.625 mg or 5mg freshly dissolved in 200l of PBS.
The durations of the injections varied in each experimental
group. An equal volume of PBS was given to control
animals.
2.2. Induction of delayed type hypersensitivity (DTH)
reaction
To induce DTH response, animals were shaved on the
dorsum (approximately 1 cm×1 cm). Sensitizationwas done
for two consecutive days by applying 25 l of 0.5% DNFB
solution gradually onto the shaved skin. DNFB (Sigma, St.
Louis, MO) solutionwas freshly prepared in acetone:olive oil
(4:1) just before use. On variable days after the last sensitization,
DTH was induced by the topical application of 20l
of 0.2% DNFB solution on the dorsal surface of the right
pinna [24]. The interval between sensitization and challenge
varied depending on the experiments. The left pinna was left
untreated or treated with vehicle only. The thickness of both
pinnae were measured with a constant-loading micrometer
(Mitutoyo, Japan) once just before sensitization (designated
as basal thickness) and for 7–10 days after challenge at the
same time of the day. The magnitude of DTH response was
expressed as the percentage of pinna swelling compared to
its basal thickness.
2.3. T lymphocyte proliferation assay
To assess the effect of mega-dose Vitamin C on na¨ıve,
act¨ıvated, and memory T cells, mice were administered
0.625 mg of Vitamin C for various days before, during, or
after sensitization with DNFB. Splenocytes were isolated
using Ficoll-Hypaque (Amersham Pharmacia Biotech, Sweden)
density gradient and incubated at 37 ◦C for 2 h to remove
adherent monocytes and macrophages, and then in
a dish coated with 10 ml of 2 g/ml anti-NK1.1 antibody
(Pharmingen, San Diego, CA) for another 2 h to eliminate
NK cells. T cells were further purified by negative selection
using Dynabeads® Mouse pan B (Dynal Biotech, Oslo, Norway)
by following the manufacturer’s instructions. Briefly,
4×107 Dynabeads were added to 107 cells in a 1.5 ml microcentrifuge
tube, mixed well, and incubated at 4 ◦C for 40 min.
The tubes were then placed in a Dynal MPC (magnetic particle
concentrator) for 2 min. Un-bound cells were collected
by washing with PBS containing 0.1% fetal bovine serum.
The purity of T cells was assessed by FACS analysis using
PE-conjugated hamster anti-CD3 antibody (Pharmingen, San
Diego, CA).
Non-specific T cell activation was determined using hamster
anti-CD3 antibody (Pharmingen, San Diego, CA) or
PMA/ionomycin (Calbiochem, San Diego, CA). Ninety sixwell
plates were coated with 100l of 1g/ml anti-CD3
antibody in each well at 4 ◦C for 24 h. T cells were plated
in coated wells (at 2×105/well), or were cultured in the
presence of 100 ng/ml PMA and 500 ng/ml ionomycin. Incubation
was carried out for 24 h in Dulbeco’s minimum
essential medium (DMEM) supplemented with 10% fetal
bovine serum (Gibco BRL, Carlsbad, CA), 100 U/ml
of penicillin and 100g/ml of streptomycin (Gibco BRL,
Carlsbad, CA) at 37 ◦C in an atmosphere supplemented
with 5% CO2. DNFB-specific T cells were also stimulated.
T cells, 2×105/well, were co-cultured in 96-well
plates with 5×105 mitomycin-treated syngeneic spleen
cells pulsed with 2,4-dinitrobenzenesulfonic acid (DNBS;
Aldrich, Milwaukee, WI), a water-soluble DNFB analogue
[25]. After 24 h, cells were pulsed with 1Ci/well of [3H]
thymidine (Amersham Pharmacia Biotech, Oslo, Norway)
for 18 h, and then harvested using a cell harvester (Skatron
Instrument, Lier, Norway) on glass-fiber filters. Radioactivity
was counted in a scintillation counter (WalK.
Noh et al. / Immunology Letters 98 (2005) 63–72 65
lac, Fort Wayne, IN) and all samples were prepared in
triplicate.
2.4. Cytokine detection
To determine the effect of a mega-dose Vitamin C on
cytokine secretion profiles, T cells were purified as described
above. T cells, 3×106/well, were plated in six-well plates
and stimulated with anti-CD3 antibody, PMA/ionomycin,
or DNBS, as described above. After 24 h incubation,
supernatants were collected. Cytokines in the cell-free
supernatants were detected using commercial quantitative
sandwich immunoassay kits for IL-2, IFN-, TNF-
(R&D
Systems, Minneapolis, MN), and IL-4 (Biosource, Nivelles,
Belgium) by following the manufacturer’s manual.
2.5. Immunization with keyhole limpet hemocyanin
(KLH) and specific antibody titration
To assess the effects of Vitamin C on humoral immune
response, we immunized mice twice, on days 1 and 21, with
an intraperitoneal injection of 100g KLH (Calbiochem, La
Jolla, CA) in 200l PBS. Control mice were injected with
PBS only. Blood samples were drawn from the orbital plexus
on days 11 and 31, sera were obtained and stored at 4 ◦C until
used.
To titrate KLH-specific antibodies in sera, 96 well-ELISA
plates (Nunc, Rochester, NY) were coated with 100l of
4g/ml KLH/well for 2 days at 4 ◦C. Plates were then briefly
washed and blocked with 1% skim milk in PBS for 1 h at
room temperature. Serum samples were diluted 1:100 in 1%
skim milk/PBS containing 0.05% Tween 20, applied to the
first row of ELISA plate, and were four-fold serially diluted.
All samples were prepared in duplicate. After incubation
for 2 h at room temperature, plates were washed with
a PBS-0.05% Tween 20 mixture, and alkaline phosphataseconjugated
secondary antibodies for each isotype were added
and incubated for 60 min at room temperature. Plates were
washed three times, and p-nitrophenyl phosphate substrate
(Amresco, Solon, OH) solution was added. OD values were
measured at 405 nm. The secondary antibodies used were
as follows; goat anti-mouse polyvalent immunoglobulins
(1:1000 diluted, Sigma), goat anti-mouse IgM, IgG1, IgG2a,
IgG2b, IgG3, and IgE antibodies (1:1000 diluted, Southern
Biotech, Birmingham, AL). KLH-specific anti-sera obtained
from other experiments were collected in a tube and used as
a standard serum in every ELISA plate. Titers are expressed
as relative to standard serum values.
2.6. Statistical analysis
Statistical analysis was performed by one-way ANOVA
with Student–Newman–Keuls post-hoc analysis using
PRISM software (GraphPad, San Diego, CA). Significance
was set at p < 0.05.
3. Results
3.1. Vitamin C pre-treatment before challenge enhances
the initial stage of DTH response, but suppresses overall
inflammation accompanying DTH response
Mice were treated intraperitoneally with a daily dose of
0.625 mg or 5mg of Vitamin C, or 200 l of PBS for 26 days,
during which sensitization (on days 4 and 5) and challenge
(on day 16) with DNFB were done. Each group consisted
of eight mice. After inducing DTH response, pinna thickness
was measured and the % increment calculated versus
basal thickness (Fig. 1A). Because, in pilot tests, ears treated
with vehicle only (acetone:olive oil) showed just slight and
transient thickness increases (data not shown), we left con-
Fig. 1. The effect of Vitamin C injected during or after the induction of
skin DTH response against DNFB. Mice were intraperitoneally injected
with daily doses of 0.625 or 5mg of Vitamin C for whole 26 days of experiment
(A) or only after challenge, (B) respectively. Sensitization was
done with DNFB on days 11 and 12. Control groups were injected with
200l of PBS. The thickness of pinna was measured after challenge as
indicated, and the percentage thickness increment vs. basal thickness was
calculated. When Vitamin C was administered all through the experimental
days (A), the pinnae of experimental groups on the first day after challenge
were dose-dependently thicker than those of the control groups (p < 0.001).
Subsequently, from the second day, this situation reversed. When Vitamin C
was administered only after challenge (B), the profile was nearly the same
as that shown by Panel A, except on the first day, when the values do not
reveal statistically significant differences. Data represent mean ear swelling
(±S.D.) of eight mice per group.
66 K. Noh et al. / Immunology Letters 98 (2005) 63–72
trol pinnae untreated (even by vehicle). Pinna thickness in all
groups peaked 2 days after challenge (32.8, 24.4, and 18.6%
increment in control, 0.625 mg-, and 5 mg-treated groups,
respectively) and then gradually decreased; control group
values were highest and those of the 5 mg-treated group
lowest. However, the values were reverse 1 day after challenge,
the initial stage of DTH response. The control group
showed the lowest increment (12.7%) and the 5 mg-treated
group showed the highest (17.9%). These differences between
the three groups were statistically significant. This pattern
was exactly the same to as that reported previously [23].
Thus, Vitamin C treatment seemed to accentuate the initial
stage of DTH response on the one hand, and to exert antiinflammatory
effects on an initiated DTH response on the
other.
Considering that intraperitoneally injected exogenous vitamin
C accumulates in several tissues of the body [23], the
observed initial accentuation of DTH response by Vitamin
C treatment could be the result of accumulated tissue Vitamin
C, or the result of any alterations of immune components
before challenge. To clarify this issue, we induced DTH response
in another set of experimental groups, which were
injected with a daily dose of 0.625 mg or 5mg of Vitamin C,
or 200l of PBS for 10 days from the day of challenge, and
then measured the pinna thickness (Fig. 1B). The overall profile
was similar to the results of the groups in Fig. 1A, except
that the increment on the first day after challenge was lowest
in the 5 mg-treated group (12.4 compared to 14.4% both in
the control and in the 0.625 mg-treated groups) even though
thiswas not statistically significant. These results suggest that
pre-treating with Vitamin C before and during sensitization
affects those immune components that induce an increase
in the early DTH response. Of course, the inflammatory response
itself seemed to be suppressed by the presence of
exogenous Vitamin C alone when the inflammation was proceeding.
3.2. Vitamin C treatment only during sensitization
enhanced the initial stage of DTH
We further divided the experimental group in Fig. 1A into
“before sensitization” and “during sensitization” subgroups.
In this case, challenge was done at 38 days, instead of at 6
days after sensitization to allow the activated immune cells to
become quiescent. Thus, four groups were given a daily dose
of 0.625 mg of Vitamin C, (1) during all experimental days
(60 days; () in Fig. 2), (2) for 7 days before sensitization
((
) in Fig. 2), (3) for 10 days during sensitization (() in
Fig. 2), or (4) for 10 days after challenge ((♦) in Fig. 2). The
control group received PBS on all experimental days (() in
Fig. 2). As is shown in Fig. 2, two groups injected during the
sensitization period showed a greater pinna thickness increment
on the first day after challenge than the other groups,
including the control group (p < 0.01). Injection of Vitamin C
before sensitization or after challenge failed to augment the
initial DTH response.
Fig. 2. The effect of Vitamin C treatment before sensitization, during sensitization,
and after challenge on DTH response was further analyzed. Mice
were intraperitoneally injected with 0.625 mg of Vitamin C for all days of
the experiment (), for 7 days before sensitization (
), for 10 days during
sensitization (), for 10 days after challenge (♦), or injected with 200 l
PBS during the experiment (). DTH response was induced with DNFB 38
days after sensitization to allow the T cells activated during sensitization
to become quiescent. Only the groups administered with Vitamin C during
sensitization (and  on the graph) showed an initial accentuation of DTH
response as shown in Fig. 1A. The data represent mean ear swelling (±S.D.)
of eight mice per group.
3.3. Vitamin C enhanced T cell proliferation
The next task was to identify which of the immune components
was most probably affected by Vitamin C during
sensitization, and responsible for the accentuation of the initial
stage of DTH response. We thought that T cells were
the most probable candidate, because T cells, especially Th1
cells, are the prime responders during sensitization and the
initiation of DTH response by antigen challenge [26]. Therefore,
we evaluated the functional differences of T cells from
mice treated and not treated with Vitamin C.
Mice were administrated 0.625 mg of Vitamin C or 200 l
of PBS (A) for 10 days without sensitization (na¨ıve T cells;
equivalent to (
) in Fig. 2), or (B) for 16 days with sensitization
at days 11 and 12 (activated T cells; equivalent to ()
in Fig. 2), or (C) for 54 days with sensitization on days 10
and 11 (memory T cells; equivalent to () in Fig. 2). At the
end of the Vitamin C administration, mice were sacrificed
and T cells were obtained as described in Section 2. Purity of
isolated T cells was over 90% as assessed by FACS analysis
(data not shown).
Na¨ıve T cells (Fig. 3A) proliferated in the presence of
anti-CD3 antibody and PMA/I, but were not proliferated by
DNBS and without stimulation (“control” in the graph). Furthermore,
the magnitude of thymidine incorporation was the
same in both the Vitamin C-treated and PBS-treated groups.
These results imply the absence of both active T cells and
DNFB-specific memory T cells. After sensitization (Fig. 3B),
T cells proliferated in the absence of any kind of proliferative
stimulus (‘control’ in the graph) implying T cell activation in
K. Noh et al. / Immunology Letters 98 (2005) 63–72 67
Fig. 3. The effect of exogenously administered mega-dose Vitamin C on
T cell proliferative activities. Mice were treated with 0.625 mg of Vitamin
C for 10 days without sensitization (A), for 16 days with sensitization on
days 11 and 12 (B), or for 54 days with sensitization on days 11 and 12 (C).
Control groups were injected with 200l of PBS. T cells were isolated and
cultured for 24 h with a DNFB-specific or non-specific stimuli (anti-CD3
antibody or PMA/I). Tritiated thymidine was pulsed for an additional 18 h
and radioactivities were measured. The filled bar () and the unfilled bar (
)
represent the Vitamin C-treated and PBS-treated groups, respectively. The
proliferation of T cells from Vitamin C-treated mice exceeded that of the
control groups only when the mice were sensitized. Values are the average
cpm±S.D. of triplicates.
vivo by sensitization. When stimulated with anti-CD3 antibody
or PMA/I, thymidine uptake was elevated more in the
Vitamin C-treated group than in the PBS-treated group. To
be noticed in these groups is that DNBS also enhanced T cell
proliferation in contrast to the na¨ıve T cell groups, implying
the presence of DNFB-specific activated T cells. T cells
from mice 42 days after sensitization (Fig. 3C) revealed similar
results to those in Fig. 3B. However, the control group
showed minimal uptake indicating the absence of activated T
cells. Meanwhile, proliferation was induced by DNBS both
in Vitamin C-treated and PBS-treated groups, which implies
the presence of DNFB-specific memory T cells. However,
the magnitude of thymidine uptake in the Vitamin C-treated
group was double that of the PBS-treated group.
Besides the status of the T cells isolated, another possible
factor that could elicit differences among groups in Fig. 3
is the duration of Vitamin C treatment (10, 16, and 54 days,
respectively). Therefore, we repeated the T cell proliferation
assay with some modification and examined the effect
of the duration of Vitamin C treatment. Mice were treated
with a daily dose of 0.625 mg Vitamin C (A) for 43 days
with sensitization on days 4 and 5, or (B) for 43 days without
sensitization, or (C) treated with Vitamin C only for 10 days
during sensitization 38 days before T cell isolation. Thymidine
uptake assays were done in the same way as in Fig. 3;
the results are shown in Fig. 4. Results for the 43 day-treated
and sensitized groups (Fig. 4A) were the same as those of
the groups in Fig. 3C, which were treated for 54 days and
sensitized. Despite long-term treatment of Vitamin (for 43
days), if sensitization was not carried out during Vitamin
C treatment (Fig. 4B, similar to Fig. 3A), T cells showed
unaltered thymidine uptake compared to the corresponding
PBS-treated group. Meanwhile, those treated with Vitamin
C only during sensitization (Fig. 4C) elicited similar results
to the sensitized 43-day Vitamin C-treated group (Fig. 4A),
suggesting that sensitization in the presence of exogenously
administered mega-dose Vitamin C results in an increase
in T cell response to both antigen-specific and non-specific
stimuli.
3.4. T cells treated with Vitamin C shifted their cytokine
secretion profiles from Th2 to Th1
The enhanced initial stage of DTH response could be regarded
as an outcome of the shift of general immune response
from Th2 to Th1 [27]. Thus, changes in the cytokine secretion
profiles of T cells could be expected. Mice were treated with
Vitamin C or PBS and T cells were isolated as in Fig. 3. After
the ex vivo stimulation of T cells for 24 h, supernatants were
collected and titers of Th1 cytokines including IL-2, TNF-

, and IFN- and the Th2 cytokine IL-4 were measured by
ELISA.
Without sensitization (left column in Fig. 5), Vitamin C
treatment did not alter the cytokine secretion profiles of T
cells versus those of PBS-treated mice, at least with respect
to the four cytokines tested. T cells not-stimulated or stimulated
with DNBS secreted no detectable cytokines. In contrast,
T cells from sensitized mice, regardless of their being
activated (middle column in Fig. 5) or memory (right column
in Fig. 5) cells, showed elevated IL-2, TNF-
, and IFN- secretions
in Vitamin C-treated mice, whereas the secretion of
IL-4 decreased.
68 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Fig. 4. The effect of the duration of Vitamin C treatment on T cell proliferative
activities. Mice were injected with 0.625 mg of Vitamin C for 43 days
with sensitization on days 4 and 5 (A), for 43 days without sensitization (B),
or for the first 10 days of the 43 days with sensitization on days 4 and 5 (C).
Control groups were injected with 200l of PBS. T cells were isolated and
cultured for 24 h with DNFB specific or non-specific stimuli (anti-CD3 antibody
or PMA/I). Tritiated thymidine was pulsed for an additional 18 h, and
radioactivities were measured. Filled bar () and unfilled bar (
) represent
Vitamin C-treated and PBS-treated groups, respectively. Even though mice
were treated with Vitamin C for 43 days, T cells show no difference in their
proliferative activity vs. those of PBS-treated mice if they were not sensitized
during Vitamin C treatment (B). In contrast, only a 10-day Vitamin C
treatment affected T cell proliferation when the treatment was done during
sensitization (C). Values are the average cpm±S.D. of triplicates.
3.5. Vitamin C decreased anti-KLH IgG1 and IgE
antibody responses in vivo
Because cytokine profiles were shifted toward Th1, it
could be predicted that humoral response would be attenuated
[27]. To verify this, we injected mice with Vitamin C
or PBS, immunized them withKLHtwice, and then measured
the serum KLH specific antibody titers by ELISA. A daily
dose of 0.625 mg Vitamin C was intraperitoneally administered;
(1) on all experimental days, or (2) during the primary
immunization for 7 days, or (3) during the secondary immunization
for 7 days. The control group was treated with PBS
on all experimental days.
The results shown in Fig. 6 are for secondary immune
sera. Whole specific titer (IgM, IgG, and IgA) was reduced
in Vitamin C-treated mice regardless of treatment time, and
this seemed to be mainly due to reduced IgG1 and IgE titer,
which are typical Th2-driven isotypes in mice. Other isotypes
were unaffected in terms of KLH specific titer.
4. Discussion
In this study, we examined the effects of Vitamin C on
immune response in Balb/c mice. Previous results that an
exogenously administered mega-dose of Vitamin C accentuated
the initial stage of DTH response [23] were reproduced
in the present experiment. When sensitization was carried
out with the administration of exogenous mega-dose Vitamin
C, the resulting in vivo activated and memory T cells
showed increased proliferative activities in response to ex
vivo antigen-specific and non-specific stimuli, and shifted
cytokine secretion profiles from Th2 to Th1. When immunizationwas
done in the presence of an exogenous mega-dose
of Vitamin C, the titer of whole antigen-specific antibodies in
secondary sera was reduced, and this reduction was mainly
attributed to decreased titers of the Th2-driven isotypes, IgG1
and IgE.
DTH response to skin sensitizer molecules such as DNFB
was used as an experimental model to evaluate cell-mediated
immune function [24]. When memory T cells against an antigen
are exposed to the same antigen again (challenge), they
are reactivated (initial stage of DTH response) to secrete cytokines
like IL-2 and IFN-, which in turn activate tissue
macrophages (cognition and activation). These events occur
within 24 h [28]. Activated macrophages again secrete inflammatory
cytokines and reactive oxygen radicals to propagate
and maintain inflammation [29].
UV irradiation [30,31] and burn stress [32] suppress DTH
response in skin by producing reactive oxygen species (ROS).
Topically or orally administered Vitamin C protects against
this suppression, probably by acting as an antioxidant. In this
experiment, we examined the effect of Vitamin C on DTH
response in normal mice, not from the aspect of protection
against DTH suppression. When injected intraperitoneally,
Vitamin C showed biphasic effect on DTH response. The
thickness of pinnae on the first day increased in a Vitamin C
dose-dependent manner. However, this thickness increment
was inversely related to the amount of Vitamin C administered
from the second day on (Fig. 1). We thought that the
Vitamin C altered T cell function and thus augmented initial,
and cognition and activation stages of DTH response, whereK.
Noh et al. / Immunology Letters 98 (2005) 63–72 69
Fig. 5. The effect of Vitamin C on cytokine secretion by T cells. Mice were treated with 0.625 mg of Vitamin C for 10 days without sensitization (left column),
for 16 days with sensitization on days 11 and 12 (middle column), and for 54 days with sensitization on days 11 and 12 (right column). Control groups were
injected with 200l of PBS. T cells were isolated and cultured for 24 h with DNFB specific or non-specific stimuli. ELISA was done on the supernatants for
the cytokines. In general, Vitamin C elevated the secretion of Th cytokines (IL-2, TNF-
, IFN-) and lowered Th2 cytokine (IL-4) secretion by T cells. Filled
bars () and unfilled bars (
) represent Vitamin C-treated and PBS-treated groups, respectively. Values are the average±S.D. of triplicates.
upon it performed a role as an anti-inflammatory agent from
the second day on.
The anti-inflammatory effects of Vitamin C have already
been suggested. It exerts these effects by scavenging ROS
that is produced by macrophages, destroys tissues and activates
macrophages and neutrophils to progress inflammation
[33,34]. It inhibits the activation of transcription factor NF-
B, which plays a critical role in the production of inflammatory
cytokines such as TNF-
, IL-1, and IL-6 [35–37].
Actually, patients with cystic fibrosis with high plasma Vitamin
C levels had lower inflammation indexes than those with
low Vitamin C levels [34]. Based on such results, we suggest
that the dose-dependent suppression of ear thickening in Vitamin
C-treated mice is due to the anti-inflammatory effect
of Vitamin C. This explanation is further supported by the
finding in Fig. 2. Challenged ears were thinner from day 2
after challenge in groups which were administeredVitamin C
during active inflammation (() and () in Fig. 2) than in the
other groups not givenVitamin C at the time of inflammation.
Even though we measured changes in ear thickness for 10
days, we focused on the first day of DTH response. To explain
the augmentation of the initial stage of DTH response,
70 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Fig. 6. The effect of Vitamin C treatment upon humoral immune response.
Mice were treated with 0.625 mg of Vitamin C for 41 days during primary
and secondary immunization ( ), for 10 days after primary immunization
( ), or for 10 days after secondary immunization (
). Control group mice
were injected with 200 l of PBS during the experiment (). Ten days after
secondary immunization, sera were obtained and ELISA was performed
for whole and each isotype of KLH-specific antibody. Titers are expressed
relative to standard serum. In all experimental groups, whole KLH-specific
antibody titers were reduced vs. the control group. Among isotypes, the titers
of IgG1 and IgE were markedly reduced in all experimental groups, and thus,
contributed to the reduction in whole antibody titer. Each group consisted of
eight mice. Values are the average±S.D. **p < 0.001 vs. control. *p < 0.01
vs. control.
we analyzed the effect of Vitamin C on T cells, the initial
effector cells in this response.We found that an exogenously
administered Vitamin C mega-dose affected T cell functions,
such as proliferative activity and cytokine secretion profiles.
Furthermore, these effects occurred only when Vitamin C
was given during the sensitization period, i.e., when na¨ıve T
cells become activated and memory T cells begin to emerge.
It is notable that the duration of Vitamin C treatment did
not significantly influence these effects. For example, though
mice were treated with Vitamin C for as much as 43 days, no
change in T cell proliferation was observed in the absence of
sensitization (Fig. 4B). On the other hand, when mice were
treated withVitamin C for only10 days, changes in T cell proliferative
activity was observed if the treatment was applied
during sensitization (Fig. 4C).
T cells obtained 43 days (Fig. 3C) and 38 days (Fig. 4A
and C) after sensitization showed minimal thymidine uptake
without any ex vivo stimulation. This implies that almost
all T cells activated through sensitization were deactivated.
Actually, in vivo activated T cells cease proliferation within
1 week of antigen challenge [38,39]. However, when nonspecifically
stimulated with anti-CD3 antibody or PMA/I,
Vitamin C-treated T cells showed more uptake of thymidine
than PBS-treated T cells, a different feature from the na¨ıve
T cells in which there was no difference of thymidine uptake
between the Vitamin C-treated and the PBS-treated groups
(Fig. 3A and B). T cells in Figs. 3 and 4A and C were composed
of not only na¨ıve T cells but memory T cells, and it
can be presumed that the difference between the Vitamin Ctreated
and the PBS-treated groups is due to the activation
of memory T cells. Indeed, when these cell compartments
were stimulated with DNBS, a vivid difference in thymidine
uptake was observed between the Vitamin C-treated and the
PBS-treated groups.We do not know whether this difference
be the result of either a difference in the number of memory
cells or a difference in the vulnerability of memory T cells to
proliferative stimuli or both. Since it has been reported that
Vitamin C inhibits the apoptosis of activated T cells [9] and
increases the number of plasma T cells in elderly patients
[40], an increased number of DNFB-specific memory T cells
in theVitamin C-treated group can be expected. However, the
increased number of memory cells cannot simply explain the
increased thymidine uptake in Vitamin C-treated T cells in
Figs. 3 and 4A and C by non-specific stimulations, which are
presumably independent of specific memory T cells. Further
studies are needed to solve this issue.
The cytokine secretion profiles of T cells were also
changed in addition to and in parallel with changes in proliferative
activities (Fig. 5); that is, the increased secretion of
Th1 cytokines (IL-2, IFN- and TNF-
) and the decreased
secretion of Th2 cytokine (IL-4). These results are similar to
the increased IL-2 and IFN- production of T cells by supplementary
Vitamin E in patients with advanced colorectal
cancer [41]. Since the elevated Th1 cytokines are involved
in cell-mediated immune responses [42,43], these results explain,
in part, the augmented initial stage of DTH response
observed in the present study.
Another point is that, since IL-4 is involved in humoral
immunity [42,43], we expected to observe reduced serum
immunoglobulin levels, and in fact this was the case. In Vitamin
C-treated groups during sensitization, total KLH-specific
antibody titers were reduced. In particular, specific IgG1 and
IgE levels were less than a tenth of those of the PBS-treated
group, markedly contributing to a reduced total specific antibody
titer. These results are in accordance with lower IL-4
secretion by Vitamin C-treated T cells, since IL-4 is known
to induce isotype switching to IgG1 and IgE [44].
Some contradictory results have been reported with respect
to the effects of Vitamin C on humoral response. Longterm
treatment of Vitamin C increased serum IgA and IgM,
but not IgG levels in humans [45]. In guinea pigs, Vitamin
C administration during immunization enhanced specific humoral
responses [46]. Many others have reported unaltered
humoral responses in Balb/c mice [8], humans [47], and in
guinea pigs [48]. These discrepancies could be the results of
differences in the Vitamin C dose and administration routes,
the co-administration of other nutrients, and species used, or
on the presence of T cell stimulation. Meanwhile, in situations
such as chronic granulomatous disease, in which we
could expect the persistent activation of T cells in vivo, longterm
treatment of Vitamin C lowered plasma Ig levels [3].
Vitamin C also reduced serum total IgE levels in asthma patients
[49].
It remains to be elucidated how the two groups treated
with Vitamin C, i.e., during primary or secondary immunization,
had the same secondary antibody secretion profiles.
Those treated only during secondary immunization would
K. Noh et al. / Immunology Letters 98 (2005) 63–72 71
have the same repertoire of memory B cells as the control
group, we would expect a higher titer of IgG1 and IgE
in this group than in the group treated during primary immunization.
However, the titers of both groups showed no
differences. The possibilities are that the isotype-switched
memory B cells during the primary response either underwent
apoptosis or isotype-switched once again during the
secondary response probably due to decreased IL-4 production
by T cells. In this experiment we obtained no data on
this issue, and a search of the literatures provided no clear
suggestions.
To summarize, mega-dose Vitamin C altered the proliferative
capacity of T cells against specific and non-specific
stimuli, and changed cytokine profiles toward Th1, and finally
resulted in an increase in the onset of DTH response and a
decrease in the total specific antibody titer in serum. This
Th1 polarization of immune response by Vitamin C has been
mentioned of its possibility [50]. What is important is that
these effects of exogenous Vitamin C occurred only when it
was administered during sensitization. Thus, we suggest that,
only activated T cells are vulnerable to changes by exogenous
mega-dose Vitamin C. It is not known whether this effect occurs
due to the intracellular accumulation of Vitamin C or
to the ligation of putative Vitamin C membrane receptors,
which have not yet been identified.
Acknowledgement
This work was supported by the Korea Science & Engineering
Foundation (KOSEF) through the Tumor Immunity
Medical Research Center (TIMRC) at Seoul National University,
College of Medicine.
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