Year : 2017 | Volume
: 9 | Issue : 1 | Page : 45--49
Neopterin as a diagnostic biomarker for diagnosis of inflammatory diseases like periodontitis
Jammula Surya Prasanna1, Chinta Sumadhura1, Parupalli Karunakar2,
1 Department of Periodontics, Panineeya Institute of Dental Sciences and Research Center, Hyderabad, Telangana, India
2 Department of Conservative and Endodontics, Panineeya Institute of Dental Sciences and Research Center, Hyderabad, Telangana, India
Jammula Surya Prasanna
Road No. 5, Kamalanagar, Dilsucknagar, Hyderabad - 500 060, Telangana
Neopterin is a catabolic product of guanosine triphosphate, a purine nucleotide and belongs to the group pteridines. When the cytokine interferon gamma stimulates the human macrophages, they synthesize the neopterin. It is an indicative of a pro-inflammatory immune status and hence serves as a cellular immune system marker. In most of the diseases, in which the cellular immune system is involved, we find that the neopterin concentrations are usually high. In the periodontal diseases, the levels of neopterin usually fluctuate which is proved by its increase in disease progression and a decrease after treatment. Periodontal diseases are characterized by enhanced macrophage infiltration to the periodontal lesion, so neopterin being a macrophage activation marker may be seen in higher levels. This review deals with neopterin and its mechanism and its use as a marker in the diagnosis of the periodontal diseases.
|How to cite this article:|
Prasanna JS, Sumadhura C, Karunakar P. Neopterin as a diagnostic biomarker for diagnosis of inflammatory diseases like periodontitis.J Oral Res Rev 2017;9:45-49
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Prasanna JS, Sumadhura C, Karunakar P. Neopterin as a diagnostic biomarker for diagnosis of inflammatory diseases like periodontitis. J Oral Res Rev [serial online] 2017 [cited 2017 Nov 17 ];9:45-49
Available from: http://www.jorr.org/text.asp?2017/9/1/45/201406
The periodontal ligament and alveolar bone are destroyed, resulting from the interaction of the host defense mechanism with the plaque microorganisms, which signifies the presence of periodontitis that is classified as an inflammatory disease. There is abundant evidence that major tissue destruction in established periodontitis lesions results from the recruitment of host cells through activation of monocytes/macrophages, lymphocytes, fibroblasts, and other cell types. Hence, the resulting infiltrate seen in periodontitis shows the presence of transmigrated mononucleus phagocytes and lymphocytes.
Majority of periodontal diseases are site specific; substances that appeared in gingival crevicular fluid (GCF) correlated with levels of the disease activity as well as tissue destruction. Commonly used clinical radiographs although can describe the present disease status, those are not adequate diagnostic aids to find initial and future periodontal destruction. If we detect and correlate disease activity in GCF or saliva before severe form using as biomarker like neopterin, might provide the current information about the tissue destruction caused and also can prevent the disease severity and tooth morbidity.
A biomarker is a feature or a facet that can be used to measure the progress of the disease or effects of the treatment. According to dictionary terms, bio means related to living beings and marker is a characteristic or factor which helps in recognizing or identifying a cell or molecule. According to NIH - biomarker is a parameter that is objectively measured and evaluated as a characteristic indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
Neopterin is a catabolic product of guanosine triphosphate (GTP), a purine nucleotide and belongs to the group pteridines. When the cytokine interferon gamma stimulates the human macrophages, they synthesize the neopterin. It is an indicative of a pro-inflammatory immune status and hence serves as a cellular immune system marker.
The neopterin levels in the body fluids such as serum and urine are increased in some of the diseases which are linked with cellular immune reaction such as the autoimmune diseases, viral infections, HIV infection, inflammatory diseases, infections by intracellular living bacteria or parasites, rejection episodes following organ transplantation, and a few malignant diseases.
In most of the diseases, in which the cellular immune system is involved, we find that the neopterin concentrations are usually high. Hence, the neopterin concentrations can be determined to measure the degree of activation of the immune system and this can be helpful as an easy and specific laboratory diagnosis.
Mechanism of the Neopterin Formation
Immunological and biological formation of neopterin
Neopterin is formed when the T-lymphocytes produce the so-called lymphokines, such as interferon gamma as different mediators when T-lymphocytes recognize foreign cells or the modified self-cells, and then, these interferons in turn stimulate the human monocytes/macrophages. These macrophages are responsible for the production as well as the release of neopterin.
Neopterin (6-D erythrotrihydroxypropyl-pterin) is a substance of low molecular mass. It is biosynthesized from GTP by an enzyme of pteridine-biosynthesis: GTP-cyclohydrolase I which cleaves the GTP into an intermediate product 7,8-dihydroneopterin-triphosphate. Unlike other cells and species, the human monocytes/macrophages only have a small constitutive activity of the biopterin-forming enzyme pyruvoyl-tetrahydropterin synthase so that exclusive synthesis and release of neopterin and 7,8-dihydroneopterin takes place, which is the cause of considerable amounts of neopterin being found only in body fluids. The biopterin derivatives are detected only in the human monocytes/macrophages or of the myelomonocytic cell line THP-1 and relevant amounts of neopterin are released. The only cytokine which produces significant amount of neopterin was identified as interferon-γ. Other cannot induce the formation of neopterin directly but can costimulate the release of neopterin which are triggered by interferon-γ. Thus, the degree of activation of the T-lymphocytes, especially of the TH-1-type cells, which are responsible for the production of interferon-γ and interleukin-2, is of great importance for neopterin production.
Therefore, the substances which can influence the T-cells activity can indirectly modify the degree of activation of monocytes or macrophages to produce and release the neopterin. Exogenous addition of interleukin-2 and interleukin-12 to peripheral mononuclear cells but also in vivo helps in activation of the T-cells and leads to an increase of neopterin expression, but no direct influence of this cytokine on neopterin production by monocytes/macrophages is seen. On contrary, cyclosporin-A which is an immunosupressant, which inhibits formation of cytokines by T-cells, can also reduce the neopterin production.
The value of neopterin monitoring is superior compared to the direct analysis of interferon-γ. Neopterin is biochemically inert and its half-life in the human organism is only due to renal excretion. The consequence of this biological half life is, the diagnostic accessibility of cytokines is limited by several circumstances; for instance, released interferon-γ is bound very quickly to target structures or becomes neutralized by soluble receptors, leading to locally formed cytokines often being unable to reach the blood circulation and no good targets for routine laboratory diagnosis being found. Beyond that, the measurement of neopterin levels does not solely reflect the effect of one single cytokine; rather, it allows determining the total effect of the immunological network and interactions on the population of monocytes/macrophages. The reflection of the multiple cooperations between immune competent cells seems to be the basis for the remarkable value of neopterin analysis as an immunodiagnostic tool.
Atypical phenylketonuria (PKU), a rare inborn metabolic defect, beside the processes that are connected with an activated cellular immune system presents with increased neopterin levels, also in which detection of neopterin is also used for differential diagnosis. Increased blood levels of phenylalanine in blood are linked with atypical PKU as it is the case in classical PKU and is diagnosed in the 1st month of life. The incidence of this inborn defect which leads to an increase of neopterin in body fluids is found to be lower than 1 out of 1 million newborns.
Neopterin derivatives and redox systems
The participation within cytotoxic mechanism of macrophages by neopterin derivate is a possibility indicated by recent findings. On one hand, the ability of monocytes and macrophages to set free reactive oxygen species (ROS) and detection of neopterin has shown a strong correlation.
Therefore, to determine the amount of immunologically induced oxidative stress, neopterin can be regarded as an indirect marker. On the other hand, the derivatives of neopterin also influence the efficacy of reactive nitrogen, oxygen, and chlorine species. Luminol chemiluminescence studies show that neopterin accelerates the formation of both reactive substances such as chloramines, HOCl, H2O2, peroxynitrite (ONOO), and scavenger entities such as 7,8-dihydroneopterin. On the basis of these results, it can be concluded that the formation of neopterin plays a crucial role in regulation of cytotoxic effect or functions of activated macrophages.
One of the prime cytotoxic reactions of activated macrophages is production of nitric oxide (NO) from arginine which is stimulated by interferon-γ through inducible NO synthase (iNOS). Although this mechanism is seen in several species, it was detected only in humans to a very minor content. Due to excess production of neopterin, physiological metabolites such as 5,6,7,8-tetrahydrobiopterin which acts as a cofactor for functioning of iNOS, is available only in minute quantity in activated human monocytes/macrophages. Thus, excess production of neopterin can compensate the deficient cytotoxic mechanism of human monocytes/macrophages. Neopterin derivatives not only enhance the effects of ONOO − but also interfere with molecular biological pathways regulated by redox-balances.
In vitro studies suggest that derivatives of neopterin have capability of regeneration, i.e., role in regulation of expression of the nuclear factor-kappa B and of the iNOS-gene as well as in degeneration, i.e., apoptosis by inhibiting the formation of erythropoietin. Recent studies depict the possible role of neopterin and 7,8-dihydroneopterin in malignant transformation of cells as they regulate the expression of proto-oncogene c-fos.
Neopterin detection in body fluids
Neopterin is considered as a low molecular weight substance which is inert biologically and chemically in body fluids; and so, it can be used for routine laboratory diagnostic measurements without any difficulty. As neopterin is eliminated by the kidney, changes in concentration of neopterin in serum are usually reflected by urine levels. In fact, there is an equal sensitivity of neopterin measurements either in serum or urine. Comparing to neopterin, 7,8-dihydroneopterin is chemically unstable and easily decomposes due to oxidation and so not suitable for routine laboratory diagnostic measurements. Proper care should be taken during sample collection and handling as it can restrict its effectiveness as a clinical diagnostic.
Specific protocol should follow during transport and storage of neopterin as it is slightly sensitive to direct sunlight. In general, dark tubes can be used for collecting samples and aluminum foils can be used for storage and transport.
Detection in protein-containing body fluids
Fortunately, both in serum and plasma, neopterin levels do not differ. Favorably, levels of neopterin determinants in serum, plasma, and other protein-containing body fluids, for example, cerebrospinal fluid, pancreatic juice or ascites, saliva, and GCF are analyzed by immunoassays (ELISA or radioimmunoassay). For a single measurement of neopterin by immunoassay, 20–100 μl of serum, plasma, or cerebrospinal fluid is essential. The samples of neopterin in serum or plasma do not require any special cooling equipment as it is stable for 3 days at room temperature. However, for storage, cooling at 4°C is adequate until 1 week, and for longer periods, the samples must be kept frozen (−20°C till 3 months). One of the main disadvantages of deep and repeated thawing-freezing cycles is depletion of neopterin levels, so it should be avoided. For measurements of neopterin concentrations in bile fluid, the samples should be diluted with physiologic NaCl solution (=0.9% acqueous NaCl), for example, 100 μl bile fluid plus 1000 μl NaCl solution.
Neopterin measurements in urine
High pressure liquid chromatography (HPLC) with Sorensen phosphate buffer (e.g., 0.015 M, pH = 6.4) on reversed phase (e.g., C18) as eluent (flow rate = 1.0 ml/min) is used for estimation of neopterin in urine samples, which allows a rapid, sensitive, and accurate prediction and meets the requirements of quality in routine laboratory investigations. 1000 μl elution buffer containing 2 g/l ethylenediaminetetraacetic acid is mixed with 100 μl of urine to dissolve all urinary sediments. Neopterin can be estimated by measuring its natural fluorescence (excitation wavelength 353 nm, emission wavelength 438 nm). Proper disinfection should be maintained by cleaning the column with a 20-min time interval gradient from buffer through water to 100% methanol, remaining on methanol for 20 min; then, gradient through water goes back to buffer (within 20 min).
Immunological methods are very similar to the HPLC measurement. However, physiological dilution of urine alters concentrations of neopterin making these less specific comparing to others. Thus, after every additional dilution step, repeated measurements of the urine samples with concentrations beyond the range of linearity should be taken.
It is advised to use the early morning urine instead of the 24 h urine to avoid laborious collection of 24 h urine. Physiological differences in urine densities and concentrations should be taken into account by calculating the ratio of neopterin concentrations versus urinary creatinine which is considered as gold standard since creatinine is excreted in relatively constant amounts during the day. When measuring neopterin concentrations using HPLC, creatinine concentrations can also be estimated in parallel by detection of its ultraviolet-absorption at 235 nm wavelength in the same chromatographic run. The quotient neopterin/creatinine is usually expressed in μmol neopterin/mol creatinine. Diurnal variation of the neopterin/creatinine concentrations is generally seen such that in the first morning, urine specimens' higher ratios (about + 20%) are observed than during the day. In general, for HPLC, 100 μl of urine is required for a single detection. Neopterin concentrations in urine specimens are stable for at least 3 days at room temperature, for storage till 1 week cooling at 4°C is sufficient, for a longer period of time samples must be kept frozen. Repeated thawing-freezing cycles have to be avoided.
Neopterin levels detected in serum or plasma do not differ. On average, the concentrations of neopterin are 5.2 + 2.5 nmol/L. Normal values and upper limits of tolerance (95th percentile) are generally depend on age of the participant. Hence, the concentrations of neopterin are classified according to three age groups. The concentration of neopterin concentrations in serum or plasma cerebrospinal fluid is somewhat higher than cerebrospinal fluid.
Which can be judged from these basic values
Upper limit of the normal (adults, 95th percentile): Serum/plasma: 8.7 nmol/LUrine, slightly age-dependent (adults, 97.5th percentile): Males: 176–229 µmol/molCreatinine; females: 208–251 µmol/mol creatinineCerebrospinal fluid: 5.5 nmol/L.
Concentrations of neopterin estimated in the first morning urine are about 1500 nmol/l, concentrations of creatinine about 12 mmol/l; thus, the gold standard value of healthy persons can be estimated at about 125 + 5 μmol neopterin/mol creatinine.
As there are variations of urinary creatinine concentrations, normal values and upper limits of tolerance of urinary neopterin concentrations in healthy persons slightly depend on age and sex.
Renal clearance of neopterin and creatinine is very similar. Therefore, even under renal impairment neopterin per creatinine ratios in urine do not impair. However, under-reduced renal excretion conditions such as uremia concentrations of neopterin in serum or plasma shows extremely high values (200 nmol/L and more). Therefore, in patients with impaired renal function, enhanced formation of neopterin by immune system activation is seen. Hence, in renal retardation conditions calculating the neopterin per creatinine ratio will be helpful in evaluating the accumulation.
Neopterin and periodontal disease
In the periodontal diseases, the levels of neopterin usually fluctuate which is proved by its increase in disease progression and a decrease after treatment. Periodontal diseases are characterized by enhanced macrophage infiltration to the periodontal lesion. Neopterin being a macrophage activation marker may be seen in higher levels. Macrophages contain peroxidase, collagenases, and several acid hydrolases and so plays a direct and important function in cell-mediated immunity. In diseased periodontal tissue, macrophage collagenase may have a significant role in collagen destruction suggesting that the levels of neopterin might be an indicator of these host mechanism leading to tissue destruction.
Collagen destruction might be caused by neopterin as it being a macrophage activation marker, leading to periodontal tissue destruction. Several recent studies estimated that neopterin also interacts with reactive oxidative stress. In periodontal destruction, monocytes/macrophages are usually activated in the course of host defense reactions, which initiates the production of reactive nitrogen species (RNS) and ROS, and this mechanism indeed causes amplification of the cytotoxic forces of ROS/RNS directed against the invading pathogens leading to further periodontal destruction.
Methods for neopterin measurements
ELISA (manually or fully automated),or neopterin estimation, each sample was centrifuged at 10,600 ×g for 10 min at 4°C. Two hundred microliters ethanol was added to 100 μl of sample to precipitate the proteins and vortexes for 1 min. The supernatant in each sample was collected for estimation after centrifugation.Reagents were allowed to reach room temperature before use:Label or mark the microtiter well strips to be used on the plate. Dilute the wash buffer with water (1:20)Pipette 20 µl of standards, controls, and samples into appropriate wells in duplicateAdd 100 µl of ready-to-use enzyme conjugate into each well. Mix gently for 5–10 s. Cover the plate and incubate for 90 min at 18–25°C temperature on orbital shaker (500 rmp) in the darkAspirate the well contents and blot the plate on absorbent paper. Immediately, wash the wells 4 times with 300 µl of ×1 wash bufferAdd 150 µl TMB substrate solution. Mix gently for 5–10 s. Cover the plate and incubate for 10 min in the dark at 18–25°C temperature Stop the reaction by adding 150 µl of stop solution to all wells at the same timed intervals as in step 5. Mix gently for 5–10 s to have uniform color distribution (blue color turns yellow)Measure the absorbance at 450 nm using an ELISA reader within 15 min HPLC Radioimmunoassay Determination of neopterin concentrations in urine: Semi-quantitative: Rapid-test (at bed-side).
With the severity of the inflammatory and destructive diseases, the neopterin levels increased in parallel in GCF suggesting it as potential indicators of periodontal inflammation and the host response. However, as there is a huge degree of variations in concentrations of neopterin from one subject to another and also in different age groups, these intersubject variations should be nullified by further studies that take disease activity into account (gingivitis sites progressing to periodontitis).
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