1 Introduction
Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms worldwide and is the most common cancer in the males and the third most common cancer in the females in India. In India, about 60,000 new cases of oral cancer are reported to occur every year with tobacco consumption being the single most important risk factor for the development of oral cancers. Bursts of reactive oxygen species in tobacco users have long been implicated as the prime form of damage brought to the genetic material leading to non-lethal mutations eventually turning out in the form of frank malignant lesions in this group of individuals (Kolanjiappan et al., 2003).

Oral squamous cell carcinoma has a much higher prevalence in the elderly age groups as compared with the younger population. This higher prevalence among the elderly population might result from an age related increase in the magnitude of the attack of free radicals including the so-called reactive oxygen and nitrogen species-ROS and RNS-causing various DNA mutations and aberrations. It might also result from an age related reduction in the body’s antioxidant defenses and/or, both (Hershkovich et al., 2007; Khanna et al., 2005).

Also, the development of cancer is multi-factorial depending on the extent of damage brought to the DNA which, in turn, is proportional to the magnitude of reactive oxygen and nitrogen stresses. This is only when this equilibrium is disturbed that the damage to the DNA is brought about and cancer evolves (Kolanjiappan et al., 2003; Bahar et al., 2007; Tandon et al., 2005; Demirbilek et al., 2007).

In plasma, free thiol groups are quantitatively the most important scavengers of the various free radicals and are known to be located largely on the various serum proteins, one amongst them being albumin. Advanced oxidation protein products, formed as a result of irreparable oxidative damage to the proteins, have been, in this regard, defined as novel and reliable markers of the irreversible oxidative damage.

Despite tremendous advances in the diagnosis and the management of oral cancers, this group of cancers is considered to be the one with the highest mortality as well morbidity rates with the diagnostic adjuncts which are used to aid an early diagnosis of oral cancers either suffering from a lack of sensitivity in the initial stages of the processes leading to frank oral cancers or suffering from a setback of not being so cost effective. In addition, biopsy, which is considered to be the gold standard in the diagnosis of oral cancers, suffers from the reliability of an appropriate site for the obtainment of the specimen to be conclusive depending largely on the area of the tumour sampled and the individual pathologist’s criteria for evaluation. The introduction of the concept of the field of cancerization has further questioned the significance of biopsy results in the approval or, rejection of the reports that come out to be confirmative of either dysplastic or, frank cancerous changes seen in the tissue. Also, clinical staging seems to correlate much better with the prognosis than the microscopic grading of the tumour.

The role of biochemical markers, on the other hand, comes out to be a convincing enough evidence of the changes taking place in the body eventually turning out to develop into frank malignant degenerations. The alteration of serum chemistry and the outpouring of the various growth factors and cytokines and tumor markers in the early enough changes leading to frank oral cancers is an added boon in the early diagnosis at a time when tissue and cell level changes are not obvious to be taken as an evidence in this regard. The role of serum advanced oxidation protein products as markers of oxidant mediated protein damage, if proven as statistically significant markers of the changes eventually leading to dysplastic changes in the body, could be helpful as an important diagnostic adjunct in the identification, possibly early identification and even more significantly, in determining the pre-disposition of the various oral pre-cancerous lesions and conditions, into their transformation to frank oral cancers as against the other subjective, invasive and not so cost-effective procedures of risk assessment, diagnosis and prognostication in oral cancers.

Hence, the present study was planned to assess the levels of serum advanced oxidation protein products in normal, healthy individuals and the individuals afflicted with the various oral pre-cancerous lesions and conditions progressing towards carcinomatous changes and the ones diagnosed with frank oral cancers.
Objectives:
1) To assess the levels of serum total protein in patients diagnosed with oral squamous cell carcinoma;
2) To assess the levels of advanced oxidation protein products in the sera of patients diagnosed with oral squamous cell carcinoma;
3) To compare the levels of serum total protein and advanced oxidation protein products in the age and sex matched normal, healthy adults and the ones afflicted with the various known oral pre-cancerous lesions and conditions including leukoplakia, lichen planus and oral submucous fibrosis, with the values obtained from the patients afflicted with oral squamous cell carcinoma.

2 Materials and methods
2.1Source of Data
The study was conducted in the Department of Oral Medicine and Radiology, Government Dental College and Research Institute, Bangalore for a period of 3 months from Jan 2010 to March 2010. The study group consisted of 60 new cases of clinically diagnosed and histologically proven, poorly differentiated oral squamous cell carcinoma, 60 patients with pre-cancerous lesions and conditions including 20 patients, each of speckled leukoplakia and erosive lichen planus and 20 patients with the well-known pre-cancerous condition, oral sub-mucous fibrosis along with 30 age and sex matched healthy controls.

2.2 Method of collection of data
None of the patients were on any treatment modality prior to inclusion in the study or, suffering from any systemic condition, especially hepatic or, renal disorders that could have affected the sera levels of proteins. Also, controls as well as the patients who were chronic alcoholics were excluded from the study to rule out the probability of derangement of liver functions that could have contributed towards the variations in sera levels of proteins.

Selected patients were explained in detail about the planned study and written informed consents were obtained. These patients were subjected to a detailed history and a thorough clinical examination using a specially prepared proforma.

2.3 Methodology
Written informed consents were obtained from the patients who were selected to be enrolled in the study.These patients were then subjected to a thorough oral prophylaxis, routine hematological examination and serum total protein and advanced oxidation protein products’ assessment.

The histopathological slides stained with haematoxylin and eosin were reviewed to include the cases of clinically diagnosed carcinoma buccal mucosa that were histologically proven as poorly differentiated, oral squamous cell carcinoma as per the biopsy results from the department of Oral Pathology, Government Dental College and Research Institute, Bangalore.

The histopathological grading of the included oral pre-cancerous lesions and conditions and frank carcinomas that were otherwise histologically proven as poorly differentiated, oral squamous cell carcinoma was further re-confirmed by the Department of General Pathology, Bangalore Medical College and Research Institute and associated Hospitals, Bangalore.

Since oral cancers are in an anatomic site that is easy to examine, the prognosis for treated lesions is supposed to be high. Although this is not usually the case as unfortunately, many health care providers are not aggressive in their clinical examination of the early, incipient lesions. That, coupled with the fact that oral cancers are usually asymptomatic, often delays the diagnosis, which adversely affects the prognosis. Moreover, a poorly differentiated or anaplastic oral squamous cell carcinoma with much cellular or nuclear polymorphism and with little or no keratin production may be so immature that it becomes difficult to identify the tissue of origin. Such a tumour often enlarges rapidly and metastasises early in its course.

The intention behind selective inclusion of histologically proven cases of poorly differentiated, oral squamous cell carcinoma was to select cases wherein the sera levels of serum total protein, albumin and advanced oxidation protein products, if, would have come to be as diagnostic markers of significance, would have provided an easy method of estimation of the response of the cases to the treatment as well as an adjunct in predicting the course, the chances to metastasize and survival rate of the patients as few studies conducted in the past have related them to aid the diagnosis and in the prediction of pathogenesis, progression, the ongoing process of transformation from the pre-cancerous stage to frank carcinomatous changes, response to treatment, their periodic assessment with the progress of treatment as well as the chances of metastasis and survival rates of the patients in relation to general body cancers.

2.4 Assessment of serum total protein and advanced oxidation protein products
Bio-chemical analysis of serum total protein and advanced oxidation protein products was done in the Department of Clinical Biochemistry, Bangalore Medical College and Research Institute and associated Hospitals, Bangalore. Assessment of serum total protein was done with the help of Biuret method while that of advanced oxidation protein products was done with the help of spectrophotometry.

2.5 Collection of blood and serum separation
For this, following an overnight fasting period, 5 ml of venous blood was taken from selected patients from the antecubital vein using a sterile disposable syringe in the sitting position between 8 A.M. and 10 A.M. The samples were allowed to clot and serum was immediately separated by ultracentrifugation taking full precautions to prevent hemolysis as an inappropriate storage or, prolonged storage as well as an early centrifugation before the settling of the formed elements of the blood and the separation of the serum after the clot formation, would have led to hemolysis on centrifugation. The supernatant was discarded and the rest of the sample was stored at -20 degrees Celsius.

2.6 Assay of serum total protein
Assessment of sera levels of total protein was done with the help of Biuret method. Sera levels of total protein were expressed as g/dL.

The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper (II) ion forms a violet-colored complex in an alkaline solution. Several variants on the test have been developed.

The biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.

In spite of its name, the reagent does not in fact contain biuret ((H₂N-CO-) _2NH). The test is so named because it also gives a positive reaction to the peptide bonds in the biuret molecule.

2.7 Procedure
An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often) followed by a few drops of aqueous copper (II) sulfate. If the solution turns purple, protein is present. 5~160 mg/mL can be determined.

2.8 Biuret reagent
The biuret reagent is made of potassium hydroxide (KOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.

2.9 Assay of advanced oxidation protein products in sera
Advanced oxidation protein products were measured by spectrophotometry. The assay was calibrated using chloramine-T and the absorbance was read at 340 nm on a microplate reader. Advanced oxidation protein products’ concentration was expressed as mmol/L of chloramine-T equivalents. Current AOPP methods suffer from poor reproducibility and accuracy due to precipitation of lipids in plasma samples. Solubilization of plasma lipids was therefore carried-out before spectrophotometric analysis of AOPP levels. It was done to prevent both loss of lipoproteins due to precipitation and overestimation as a result of light scattering.

Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.

The sequence of events in a modern spectrophotometer is as follows:
1) The light source is imaged upon the sample.
2) A fraction of the light is transmitted or reflected from the sample;
3) The light from the sample is imaged upon the entrance slit of the monochromator;
4) The monochromator separates the wavelengths of light and focuses each of them onto the photodetector sequentially.

Many older spectrophotometers must be calibrated by a procedure known as "zeroing." The absorbancy of a reference substance is set as a baseline value, so the absorbancies of all other substances are recorded relative to the initial "zeroed" substance. The spectrophotometer then displays % absorbancy (the amount of light absorbed relative to the initial substance).

2.10 Method of Statistical Analysis
The following methods of statistical analysis were used in the study. The results were averaged (mean + standard deviation) for continuous data and number and percentage for dichotomous data were presented in the Tables and Graphs. One way analyses of variance (Anova) were used to test the difference between groups.

2.11 One way analysis of variance (Anova)
Analysis of Variance is a technique by which the total variation is split into two parts, one, between the groups and the other, within the groups. If the F value is found to be significant, there is a significant difference between group means.  In case, if the F value is not significant, it indicates that there is no significant difference between the groups and stops the analysis at this stage. To find out which of the two groups means is significantly different, post-hoc test of Bonferronitest is used. In case of F value that is not significant, it indicates that there is no significant difference between the groups and stops the analysis at this stage and does not use Bonferronitest. Comparison of two variance Sa² and Sb², estimated for two groups Na and Nb subjects respectively uses F test as:

 With N_a-1 and N_b-1 degrees of freedom
In above test, P values less than 0.05 were taken to be statistically significant. The normality of data was checked using Kolmogorov-Smirnov and Shapiro- Wilk tests for significance before the statistical analysis was performed.

3 Results
The study revealed variations in sera levels of advanced oxidation protein products to be statistically significant (p<0.001). Sera levels of total protein showed extensive variations in the study and the results were largely inconclusive and statistically insignificant.

While the mean values of serum total protein were much the same in controls (8.11+/-1.54 g/dL) as against pre-cancerous lesions and conditions (8.65+/-3 g/dL) and oral squamous cell carcinoma (7.49 +/-2.72 g/dL), there were observed great variations in the minimum (1.8 g/dL) to maximum (19.3 g/dL) values for pre- cancerous lesions and conditions to a minimum (0.7 g/dL) to maximum (18.2 g/dL) for frank oral cancers. The p-value also came out to be statistically insignificant, 0.061 (Table 1; Figure 1).
 

marked variations in controls (0.08+/-0.03 mmol/L) and as the patients progressed from oral pre-cancerous lesions and conditions (0.37+/-0.09 mmol/L) to frank oral cancers (0.42+/-0.21 mmol/L) with a p-value of <0.001. Serum levels of advanced oxidation protein products ranged from being 0.08 mmol/L minimum to a maximum of 0.53 mmol/L in patients with oral pre-cancerous lesions and conditions to 0.14 mmol/L minimum to as high as 0.92 mmol/L maximum in patients afflicted with oral cancers (Table 2; Figure 2).
 

4 Discussion
Oxidative stress is a general term used to describe the steady state level of oxidative damage in a cell, tissue or, organ, caused by the reactive oxygen species. This damage can affect a specific molecule or, the organism as a whole (Soejima et al., 2004). Reactive oxygen species such as free radicals and peroxides represent a class of molecules that are derived from the metabolism of oxygen and exist inherently in all aerobic organisms. Most reactive oxygen species are generated from the endogenous sources as byproducts of normal and essential metabolic reactions such as energy generation from mitochondria or, the detoxification reactions involving the hepatic microsomal enzyme system. Exogenous sources include exposure to cigarette smoke, environmental pollutants such as emission from the automobiles and industries, consumption of alcohol in excess, asbestos, and exposure to ionizing radiation in addition to the plethora of the bacterial, fungal and viral infections.

The role of oxygen free radicals in the initiation, promotion and progression of carcinogenesis and the protective role of anti-oxidant defenses has been the subject of much speculation in the recent past with conflicting reports in the literature (Kolanjiappan et al., 2003). In recent years, increasing experimental and clinical data have provided compelling evidence for the involvement of oxidative stress in a large number of pathological states including cancers (Nicholson et al., 2000). The determinants of oxidative stress are regulated by an individual’s unique hereditary factors as well as environment and characteristic lifestyle. Unfortunately, under the present day life style conditions, many people run an abnormally high level of oxidative stress that could increase their probability of early incidence of decline in optimum body functions and lead to a number of pathologies including cancers (Nicholson et al., 2000; Elango et al., 2006; Halliwell and Gutteridge, 1999; Dalle-Donne et al., 2006; Halliwell and Whiteman, 2004, Lemineur et al., 2006; Capeillère-Blandin et al., 2004; Halliwell, 2001; Kaneda et al., 2002; Kalousova et al., 2002).

 
Most free radicals are highly reactive and short lived (Kolanjiappan et al., 2003; Khanna et al., 2005; Titus et al., 2004). Sun has proposed that free radicals are involved in both the initiation and promotion of multistage carcinogenesis. These free radicals have been shown to cause DNA damage, activate procarcinogens and alter the cellular anti-oxidant defense mechanisms. To counteract these lethal effects, normal living cells have multiple antioxidant defense mechanisms acting in a cascade manner (Khanna et al., 2005; Servettaz  et al., 2007).

Oxidative stress is however not always detrimental. Selective oxidative stress, sometimes, is desirable and can even possess therapeutic significance. Certain drugs including the class of anti-malarials like chloroquin, quinine, mefloquine, primaquin and  artemisinin, antibiotics like ciprofloxacin, anti-cancers including bleomycin, calcitriol and iron chelators are included in such category of drugs that utilize oxidative stress for deriving therapeutic advantage (Engel and Evens, 2006).

Plasma is known to contain a wide range of important antioxidants including albumin, ascorbic acid and uric acid. In contrast, concentrations of enzymes such as super-oxide dismutase, reduced glutathione and catalase, all of which are known to be important intracellular antioxidants, are low in plasma. While ascorbate is an important extra-cellular antioxidant, albumin via its thiol groups, provides quantitatively almost ten folds greater antioxidant protection against the various reactive oxygen and nitrogen species held responsible for the genetic damage eventually leading to the development of cancers (Kolanjiappan et al., 2003; Khanna et al., 2005; Demirbilek et al., 2007; Nicholson et al., 2000; Titus et al., 2004; Chuang et al., 2006; Ihara et al., 2004; Himmelfarb and McMonagle, 2001).

The analysis of changes in serum total protein in malignancy is, in itself, a means of studying abnormality in the protein metabolism in this condition. Until recently, radical induced damage to proteins was considered to be mainly a chain-terminating process and it was thought that the products of the damage produced on the protein ,for example, protein scission, cross-linking, chemical modification of side chains, were relatively inert; these damaged materials are thought to be subsequently degraded by intra-and extra- cellular enzymes. It has been recently demonstrated that two types of material ie. Protein-bound reducing moieties subsequently identified mainly as 3, 4-dihydroxy phenylalanine, and protein peroxides, are capable of initiating further chemical reactions. Thus, the protein bound reducing moieties have been shown to be able to reduce transition metals resulting in redox cycling of these species which are normally present solely in their oxidized states while protein peroxides, on the other hand, have been demonstrated to consume important cellular antioxidants/reductants such as ascorbates and glutathione via redox reactions (Kolanjiappan et al., 2003; Tsantoulis et al., 2007; Enwonwu and Meeks, 1995; Rasnick and Duesberg, 1999; van Kempen et al., 2006; James and Hay, 1968; Iwao et al., 2006). For similar reasons, serum total protein, in our study, came out to be statistically insignificant implying the role of the several complex factors in protein metabolism in cancer patients.

There are several different methods described in the literature for the estimation of proteins in the sera. The biuret test is one such chemical test used for detecting the presence of peptide bonds. This assay is based on copper ions binding to peptide bonds of protein under alkaline conditions to give a violet or purple colour. The intensity of the charge transfer absorption bond resulting from the Cu-protein complex is linearly proportional to the mass of protein present in the solution. The Biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The chromophore or light-absorbing centre seems to be a complex between the peptide backbone and cupric ions. The intensity of the colour, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. Several variants on the test have been developed in the recent times to increase its sensitivity and its application for the estimation of proteins.

Despite its name, the reagent does not in fact contain biuret ((H₂N-CO-) _2NH). The test is so named because it also gives a positive reaction to the peptide-like bonds in the biuret molecule. The biuret reagent is made of potassium hydroxide (KOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.

Biuret method was used for the estimation of total protein and albumin in the study because it is considered as one of the simplest methods for protein estimation. This method is sensitive to the amino acid composition of the protein. Its sensitivity is moderately constant from protein to protein and because of its simple procedure and quick result, it is used to estimate protein in crude extract over a large range of concentration.

Also, as stated before, cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells and this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and various other pathologies, even cancers (Elango et al., 2006; Kalousova et al., 2002; Titus et al., 2004; Zoellner et al., 1996; Armstrong et al., 1998; Barle et al., 2006).

Advanced oxidant protein products, first described by Witko-Sarsat et al. (1996), further have been hypothesized to activate the endothelial cells and to a lesser extent, fibroblasts to generate reactive oxygen species (Demirbilek et al., 2007; Witko-Sarsat et al., 1998; Witko-Sarsat et al., 1999). Furthermore, advanced oxidation protein products generated by different oxidation patterns lead to the production of either NO or, H₂O₂ suggesting their role in the generation of different types of reactive oxygen species that set a cascade of reactions with a potential to damage cellular micro-molecules, eventually turning out into frank oral cancers (Iwao et al., 2006; Patel et al., 2008).

The level of advanced oxidation protein products in our study ranged from a minimum of 0.08 mmol/L to 0.53 mmol/L in patients diagnosed with the numerous potentially malignant pre-malignant/pre-cancerous lesions and conditions to as high as 0.92 mmol/L in patients diagnosed with oral squamous cell carcinoma while being negligible in the controls, ranging from a minimum of 0.04 mmol/L to 0.15 mmol/L seen as the upper limit.

5 Conclusion
Reactive oxygen and nitrogen stresses have long been implicated in the genesis of oral cancers. There is enough literature available that shows convincing evidence in the use of anti-oxidants as chemo- preventive agents to stop the conversion of the various oral pre-cancerous lesions and conditions into frank oral cancers. The results obtained emphasize the need for more studies to be conducted for the assessment of sera levels of total protein and advanced oxidation protein products to accept the utility of these parameters as relevant diagnostic adjuncts and to assess their role in the pathogenesis and their impact on the prognosis of oral squamous cell carcinomas providing a scientific ground for the use of  the various chemopreventive strategies in controlling damage at genetic and molecular levels to prevent the ongoing transition of the various oral pre-cancerous lesions and conditions to frank oral cancers.

Recommendations
The results obtained emphasize the need for more studies to be conducted for the assessment of sera levels of total protein and advanced oxidation protein products to accept the utility of these parameters as relevant diagnostic adjuncts and to assess their role in the pathogenesis and their impact on the prognosis of oral squamous cell carcinomas providing a scientific ground for the use of the various chemo-preventive strategies in controlling damage at genetic and molecular levels to prevent the ongoing transition of the various oral pre-cancerous lesions and conditions to frank oral cancers .

Ethical declaration
The study has been approved by the ethical committee appointed by the Government Dental College and Research Institute, Bangalore, where the study was carried out, and Bangalore Medical College and Research Institute, Bangalore and has been performed in accordance with the ethical standards laid down in the 1975 declaration of Helsinki and its later amen- dments in 2000 after a written informed consent from the patients for their inclusion in the study. Details that might disclose the identity of the patient have been omitted.

Acknowledgement
We thank all the people who directly and indirectly contributed for the study as the study required intense efforts from the people outside our Department including the cancer wards and the Department of Clinical Biochemistry, Bangalore Medical College and Research Institute and Associated Hospitals. We feel highly indebted to Dr.Vijayalakshmi K.R., Assistant Professor, Department of Oral Medicine and Radiology, Government Dental College and Research Institute, Bangalore for her continuous support and careful revision of the manuscript.

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