toxic epidermal necrolysis ( ten ) and stevens  johnson syndrome ( sjs ) are severe cutaneous drug reactions of unknown mechanism . toxic epidermal necrolysis represents the most severe variant of a disease spectrum that consists of bullous erythema multiforme ( em ) and sjs and has a mortality rate of 3040% . keratinocyte apoptosis has now been established as a major feature in ten and sjs , with cell death resulting in detachment of the epidermis . though studies have identified molecules such as the fas ligand ( fasl ) and its receptor as well as tumor necrosis factor - alpha ( tnf- ) to be implicated in the process , the triggering event inducing apoptosis is currently unknown . fasl is a transmembrane protein from the tnf family that is expressed on the surface of cytotoxic t cells , nk cells , immune privileged cells of the testes and eye , and keratinocytes . on activation of cytotoxic t cells , fasl is expressed on their surface and binds to its receptor on target cells , which activates intracellular caspases , leading to the controlled destruction of the target cell . other cytokines and molecules , such as tnf- and nitric oxide ( no ) , have been implicated in the apoptosis of ten . tumor necrosis factor- has been shown to activate the  death receptor  tnf - r1 , causing caspase activation and cell death . the role of no in apoptosis is thought to be due to stimulation of the activity of caspases through the action of p53 . the combination of tnf- , no , and the resultant elevation of reactive oxygen species may also contribute to resultant oxidative stress and the disruption of the intracellular structures leading on to apoptosis . ten is initiated either by noncovalent , direct interaction of a drug with a specific mhc i allotype or by covalent binding of a drug metabolite to a cellular peptide , forming an immunogenic molecule . cd8 cells , activated by keratinocytes and apcs expressing specific mhc i and antigen , release inf- , causing the activation of macrophages and keratinocytes and perforin / granzyme b and granulysin . expression of glutathione - s - transferase pi ( gst - pi ) , which is induced by oxidative stress , was found to be elevated in keratinocytes of ten patients . oxygen - free radicals have also been shown to induce apoptosis in keratinocytes in culture . it is hypothesized that oxidative and nitrosative stress caused by drug metabolism is a trigger for keratinocyte apoptosis in the epidermis seen in ten and sjs . this hypothesis was tested in patients with ten and sjs by the pursuit of the following specific aims . to estimate oxidative damage in the serum in patients with ten and sjsto examine the role of no in mediating epidermal damage in ten and sjs . 
  to estimate oxidative damage in the serum in patients with ten and sjs to examine the role of no in mediating epidermal damage in ten and sjs a prospective study was conducted in a tertiary care center between january 2006 and february 2010 . patients with ten , sjs and sjs  ten overlap were included in the study group after obtaining an informed consent . to ascertain whether oxidative stress was unique to ten , patients with a maculopapular drug rash without detachment of skin were included in the control group 1 ( drug exposed ) . patients undergoing minor dermatosurgical procedures and without a drug - induced rash were included in control group 2 ( drug unexposed ) and their serum was used for biochemical analysis . patients who did not wish to be included in the study and patients with human immunodeficiency viral infection , hepatitis b and hepatitis c were excluded from the study as these infections are known to induce oxidative stress in tissue . protein carbonyls and lipid peroxidation parameters such as malondialdehyde and conjugated dienes and oxidative stress markers , levels of nitrate and nitrite , stable end products of no were estimated in the serum from all patients . the alterations in protein carbonyl content and lipid peroxidation parameters , serum nitrite / nitrate levels , active metabolites of no were compared between the groups . measurement of protein carbonyls in serum : samples were treated with 10 mm 2 - 4 dinitrophenyl hydrazine dissolved in 2n hcl , followed by incubation at room temperature for 1 hour . trichloroacetic acid ( 10% final concentration ) was then added , followed by centrifugation to pellet the precipitated protein . to this pellet , an equal volume of 1:1 ( v / v ) ethyl acetate : ethanol was added . following centrifugation , the pellet was collected and dissolved in 1 ml of 6 m guanidine hcl . absorbance at 366 nm was then measured and was expressed as nmoles / ml serum . measurement of malondialdehyde and conjugated dienes : for measurement of malondialdehyde , samples ( 200ul ) were treated with 1.2 ml of 5% tca and 0.4ml of 0.8% thiobarbituric acid . the absorbance was then read at 532 nm , and amount of malondialdehyde formed was calculated from a standard curve prepared using 1 , 19 , 3 , 39 tetramethoxypropane and was expressed as nmoles / ml serum . for conjugated diene measurement , total lipids were extracted from serum , dissolved in 1 ml heptane , and the absorbance was read at 233 nm in a spectrophotometer . concentration was calculated using a molar absorption coefficient of 2.52  104 and expressed as  mol / ml serum . measurement of nitrite / nitrate : for measurement of nitrite , nitrate in the samples was first reduced to nitrite , which was then measured by the griess reaction . reduction of nitrate to nitrite was carried out using a copper  cadmium alloy , which was prepared by mixing molten copper with cadmium in the ratio 1:9 . filings of the alloy were then prepared , followed by activation as follows : the copper  cadmium alloy filings were washed twice with 100 ml of de - ionized water , followed by two washes with 0.5n hcl . the activated filings were then washed with 0.1n hcl and stored in 0.1n hcl at 28c until use . for analysis of nitrate / nitrite , samples were incubated with the alloy filings in carbonate buffer for 1 h at room temperature with shaking . the reaction was stopped by addition of 0.35 m naoh and 120 mm znso4 solution , followed by vortexing . after standing for 10 minutes aliquots of the clear supernatant are treated with 1% sulfanilamide and 0.1% n - naphthylethylenediamine . after 10 min , the optical density was read at 545 nm in a spectrophotometer . two - group comparison with the non - parametric mann fourteen patients with sjs / ten ( study group ) and 10 patients with a maculopapular drug rash ( control group 1 ) were included in the study . four patients from the study group , two patients from control group 1 and three patients from control group 2 were excluded from the study as their blood samples were hemolysed . the study patients consisted of 10 patients in the sjs  ten group ( study group ) , 8 patients in control group 1 and 7 patients in control group 2 . majority of patients in the study group ( m : f 1:3 ) as well as in the control group 1 ( m : f , 3:4 ) were females . more than one drug was implicated in 4/10 patients in group 1 and 3/8 patients in group 2 . carbamazepine was one of the most common drug implicated in 4/10 of study group and 2/8 of the control group patients . the drugs implicated in causing the ten , sjs or drug rash is listed in table 1 . scorten of 0 , 1 and 3 at admission were seen in 2 , 6 and 2 patients of the study group , respectively . flow chart of case selection drugs implicated in the study and control group the mean values of protein carbonyls , conjugated dienes , malondialdehyde and nitrate levels of all the patients included in the study is shown in table 2 . the mean value of malondialdehyde in the control group 2 was elevated as compared to the study group and control group 1 . whitney u test showed that there is no statistically significant difference between the serum conjugated diene , nitrates , malondialdehyde and protein carbonyls between the ten group and the control groups . toxic epidermal necrolysis and sjs are characterized by the rapid onset of keratinocyte cell death by apoptosis , a process that results in the separation of the epidermis from the dermis . an immune response to defective drug metabolism has been implicated in the process and studies examining apoptotic pathways in this context have identified interaction between fas and fasl playing a role . other studies have shown soluble fasl ( sfasl ) secreted by peripheral blood mononuclear cells ( pbmcs ) and perforin and granzyme as playing a major role in apoptosis in sjs and ten . a recent study has shown secretory granulysin produced by the fully activated cytotoxic t lymphocytes ( ctls ) , natural killer ( nk ) cells and natural killer t ( nk t ) cells leads to widespread apoptosis in sjs  ten . various new markers demonstrated in ten include serum granulysin , high - mobility group box protein 1 ( hmgb1 ) , alpha defensins 13 , thymus and activation regulated chemokine ( tarc ) , bcl-2 expression in dermal infiltrate and glutathione - s - transferase pi expression in keratinocytes . another molecule which could play a role in apoptosis is no whose role in cellular signaling is now well recognized . low concentrations of no generally have a beneficial effect in cellular systems , protecting against cell death and high no concentrations can damage cellular components and induce apoptosis . the effects of no on induction of apoptosis in keratinocytes have also been shown to be influenced by free radicals such as superoxide . toxic effects of no are generally mediated through formation of reactive nitrogen species such as peroxynitrite , which can be formed in a redox environment with high no concentrations . no is produced by one of the three isoenzymes of nitric oxide synthase ( nos ) . activity of inducible form of nos ( inos ) is independent of the calcium concentration and has the capacity to produce large quantities of no . expression of inos has been demonstrated in the lesional skin of patients with sjs and ten . it has been shown that activated t cells secrete high amounts of tnf- and ifn- , and that both cytokines lead to increased expression and activity of keratinocyte inos . the resulting increase in no significantly upregulates keratinocyte fasl expression , resulting in fas- and caspase-8-mediated keratinocyte cell death . it was found that activity of the anti - oxidant enzyme superoxide dismutase ( sod ) was increased in all types of non - immediate reactions ( including urticaria , maculopapular exanthema and toxic epidermal necrolysis ) , accompanied by elevations in lipid peroxidation in urticaria and maculopapular exanthema , and carbonyl groups in all types of reactions . however , in the present study , the serum values of protein carbonyls , malondialdehyde , conjugated diene and nitrates were not significantly increased in the study group when compared to the controls . the biochemical values of patients who have received steroids were not different from the values of other patients ; hence , exposure to steroids did not seem to have altered the values . the malondialdehyde levels in the control group were higher than the study group and control group 1 . studies have shown oxidative stress markers can be elevated both during surgery and in the immediate post - operative period . verma et al . in their study on oxidative stress markers in cutaneous adverse drug reaction found that the mean malondialdehyde levels were raised in the study group as compared to the control group . our study did not show elevated levels of protein carbonyl , conjugated dienes , malondialdehyde and nitrates in patients with sjs and ten . as this was a pilot study with less number of patients , a definitive conclusion could not be drawn from the results of this study . tissue levels of protein carbonyls , malondialdehyde , conjugated dienes and nitrates would have given a better understanding of oxidative stress . since a recent study demonstrated that treatment with the anti - oxidant n - acetyl cysteine , or its combination with infliximab , did not appear to reverse the evolving ten process the role of oxidative stress in conditions such as ten may be complex and need further detailed evaluation . in our study population , oxidative stress markers were not elevated , hence a role of antioxidants in the treatment of ten can not be proposed . studies with a larger group of patients examining alterations in skin biopsies may be required to ascertain the role of oxidative and nitrosative stress in pathophysiology of ten and sjs . immunopathogenesis of ten is complex , the end result of which is apoptosis of keratinocyte . our study could not demonstrate an elevation of oxidative stress markers in patients with ten and sjs as compared to the control population . in our pilot study , serum oxidative stress markers in patients with sjs and ten were not elevated as compared to the controls . other pathogenic markers or molecules need to be studied as an inducer of keratinocyte apoptosis in ten .
background : oxidative and nitrosative stress caused by drug metabolism may be a trigger for keratinocyte apoptosis in the epidermis seen in toxic epidermal necrolysis ( ten ) and stevens johnson syndrome ( sjs).aims : to estimate oxidative damage in the serum and to examine the role of nitric oxide in mediating epidermal damage in patients with ten and sjs.materials and methods : a prospective study was conducted among ten and sjs patients and controls in a tertiary care center between january 2006 and february 2010 . 
 patients with a maculopapular drug rash without detachment of skin constituted the control group 1 ( drug exposed ) . 
 patients without a drug rash constituted the control group 2 ( drug unexposed ) . 
 the serum values of protein carbonyls , malondialdehyde , conjugated diene and nitrates were measured . 
 two - group comparison with the non - parametric mann 
 whitney u test was used . 
 significance of differences if any was established using pearson 's chi - square test.results:ten patients in the sjs - ten group ( study group ) , 8 patients in control group 1 and 7 patients in control group 2 were included . 
 more than one drug was implicated in 4/10 patients in group 1 and 3/8 patients in group 2 . 
 scorten of 0 , 1 and 3 at admission were seen in 2 , 6 and 2 patients , respectively . 
 the serum values of protein carbonyls , malondialdehyde , conjugated diene and nitrates were not significantly increased in the study group when compared to the controls.conclusions:there was no elevation of oxidative stress markers in patients with ten and sjs as compared to the control population .