IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Elevated Levels of Sialic Acid and Lipid-Associated Sialic Acid in Plasma of Rheumatoid Arthritis Patients N. S. Hamad Departme nt of Chemistry, College of Science, Unive rsity of Sulaimani Abstract The objective of this st udy is to evaluate plasma levels of total Sialic acid TSA and Lip id –associated Sialic acid LSA as a marker of Rheumatoid Art hritis AR. Plasma Sialic acid is known as a parameter of inflammation. In the p resent study , in order to exp lore the p otential role of sialic acid in arthritis rheumatoid, p lasma sialic acid levels, p lasma LSA and total p rotein in p atients with arthritis rheumatoid were measured. A total 40 p atients were comp ared with 40 healthy control subjects. Plasma TSA, LSA and TP level were determined sp ectrop hotometrically in p lasma samples. Plasma Sialic acid levels were significantly increased in RA (88.48±14.15 mg/dl, P<0.05) and LSA level were significantly increased in RA (26.37±2.25 mg/dl, p <0.05). By contrast there wasn’t any significant elevation of TP in RA p atients, comp ared with healthy control (TSA,53.31±7.58 mg/dl, P<0.05), (LSA,18.46±1.45 mg/dl) and (TP, 7.39±0.34 g/dl). Key word: Sialic acid, Lip id- associated Sialic acid. Introduction Sialic acid (N-acety lneuraminic acid NANA)are acety lated derivatives of neuraminic acid .They are att ached to non reducing residues of the carbohy drate chains of glycoproteins and glycolipids. The suggested biological functions of sialic acid are as following:(a)st abilizing the conformation of glycoproteins and cellular membranes;(b)assisting in cell to cell recognition and interaction ; (c)contributing to membrane transp ort;(d)affecting the function of membrane receptors by p roviding binding sites for ligand ;(e)influencing the function st ability and survival of blood glycoproteins ;and (f) regulating the p ermeability of the basement membrane of glomeruli[1].Sialic acid concentration varies p hy siologically with age, but its level may also be influenced by such acondition as inflammation [2].Neop last ic tumors or inborn genetic disorder which cause abnormal sialic acid metabolism[3].Sialic acid and its derivatives were used in the treatments of several diseases, including neuropathic and inflammatory diseases as well as certain tumors[4].Lip id-associated sialic acid(LSA)is a useful adjunct in the management of a variety of malignancies [5]. Elevation in blood LSA level was rep orted in p atients with mammary(63%),gastroenteric(65%),p ulmonary(79%)and ovarian(94%) neoplasms as well as those with leukemia(91%),lymphoma(87%),melanoma(84%),and Hodgkin disease(91%).[6 – 11].Sialic acid level do not app ear to be a good marker for discriminating malignant from non malignant disease of the Lung[12]. Higher levels of total sialic acid were found in women with metabolic gestation sy ndrome [ 13]and during p eriodontal disease [14]. ESR is the most commonly p erformed laboratory test for inflammation in rheumatic disorder reactive protein,transferrin, ceruloplasmin,albumin,α1-antitry p sin were similarly used[15, 16]. .The p resent st udy was undertaken to evaluate the utility of TSA and LSA as an index to measure the disease activity and inflammation in Rheumatoid Art hritis(RA).TSA, LSA and IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Total Protein TP were studied in patients with Rheumatoid Art hritis .These were comp ared with normal healthy controls. Materials and Methods Chemicals: Standard solution for sialic acid 500micro gram/ml concentration was p repared by dissolving 50 mg of st andard N-acety lneuraminic acid in 100ml of distilled water, and on the day of determination, the stock solution was diluted with p hosp hate buffer saline at p H 7.4 to give the following st andard solutions(5,10,15,20,25,30 micro gram/ml)for calibration curve measurement. Patie ns: Plasma for the measure ment of TSA , LSA and TP levels was obt ained from 40 p atients with rheumatoid arthritis of subjects followed at the central lab in Su laimania for the p eriod from Dec. 2007 to Ap r.2008.The diagnosis of t his case was sp ecified by consultants. Plasma preparation: Venous blood was collected from fast ing p atients in the early morning. The samples were taken in tubes containing anticoagulant, their p lasma were sep arated and used immediately ,otherwise , plasma samp les were st ored at [-15]until analysis. Biochemical methods: 1-Total s ialic ac id A-Principle The principle of this method dep ends on the formation of chromo gen from the add ition of resorcinol rea gent in to the test tube. The chromogen formed was e xtracted by butyl acetate/methanol r eagent and measured at 580 nm[17]. B-Reagents Reagent I: Resorcinol st ock: made up by dissolving 2 gm of resorcinol in 100 ml of deionized water. This reagent is stable for many months in the refrigerator. Reagent II: Resorcinol HCL: 10 ml of the stock solution is added to the mixture of 80 ml of concentrated HCL and 0.25 ml of 0.1M CuSO4. The volume is then made up to 100ml with deionized water. This should be p repared at least four hours before use and st able for two weeks in the refrigerator. Reagent III: buty l acetate/methanol: 85ml of buty l acetate is added to 15ml of methanol. C-Procedure: The reaction was p erformed in 180×150mm Pyrex test tube labeled as test, standard and blank, into which the following reagents were pipettes as follows: Blank µl Standard µl Test µl Reagent - - 20 sample 980 980 980 D.W - 20 - standard 1000 1000 1000 Reagent II 2000 2000 2 000 Reagent III IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 The tubes were capp ed with glass bulbs and heated for 15 min in a boiling water bath (100C) and then cooled in tap water bath or ice for 10 min.Vortexes and centrifuged for 10min at 3000 rp m, the extracted chromophore was read at 580nm. LSA dete rmination: LSA was measured according to the method described by Katop odis and his co-worker [18, 19] Fifty µl p lasma aliquots were p laced in screw-cap p ed tubes,3ml of cold of (4C)chloroform/methanol(2:1v/v)mixture was added to each tube for total lip id extraction, the tubes were then cap p ed and vortexes for 30 secons,0.5ml of cold water was added to each tube and the tubes were centrifuged for 5 min. at 2500 rp m at room temp. The up p er p hase (aqueous Lay er containing LSA) was transferred to another screw- cap p ed tube and 50micro ml of p hosp hotungst ic acid (1g/ml) was added to each tube. The tubes were vortexes again and allowed to st and at room temp . for 5min.Then the tubes were centrifuged at room temp for 5min.at 2500rp m.After that, the sup ernatants were decanted and the remaining p ellets were redissolved in 1ml of water at 37 ہ C by vigorously vortexing them for 1min and sialic acid content was determined as mentioned for TSA. TP dete rmination: Colorimetric method was described by Gornall etal[20, 21]. The p eptide bonds of p rotein react with Cu 2+ in alkaline solution to form a colored comp lex whose absorbance is p rop ortional to the concentration of the total p rotein in the sp ecimen, and was measured at 550 nm. The biuret reagent contains sodium p otassium tartrate to comp lex cupric ions and maintain their solubility in alkalin solution. Re agents: Reagent I: Sodium hy droxide 370 mmo l/L+ Na-K T artrate 10 mmol/L + Potassium iodide 3 mmol/L +Copp er sulfate 3 mmol/L Standard solution –Bovine Albumin 6 gm/d l. Procedure: Let st and reagent and sp ecimens were left at room temp erature Test µl Standard µl Blank µl Pipette into test tube 1000 1000 1000 Reagent 20 standard 20 specimen 20 D.W The above components in the tubes were mixed, left to stand for 10 minutes at room temp erature. Record absorbance's at 550nm against reagent blank. Calculation Result = Abs(Assay)÷Abs(standard) ×standard conc. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Results The levels of p lasma sialic acid TSA indicate significant st atist ical differences in the Rheumatoid arthritis p atients group comp ared to the control group (P<0.05).There is also a significant st atist ical difference (P<0.05) with the resp ect to the levels of p lasma-associated sialic acid between the control group and rheumatoid arthritis p atients group (table-1)(fig- 3,fig-4).Figs (1 and 2) show the p ercent of TSA,LSA and TP in rheumatoid arthritis and control group . TP not changed in this rheumatoid Fig-5. Table (1) also shows the M ean+- SD, variance , P-value, Confidence interval ,selectivity and sensitivity for all tests(T SA,LSA and TP). The correlation coefficient equals -0.27, indicating a relatively weak relationship between the variables TSA and LSA (Fig -6-a). The correlation coefficient equals 0.25,indicating a relatively weak relationship between the TSA and TP(Fig-6-b),where the correlation coefficient equals 0.10 ,indicating a relatively weak relationship between the variables LSA and TP (Fig-6-c).The p resent st udy shows that the magnitude of the selectivity varies between (60% for TP )for p atients with RA ,while equals 82.5% for TSA and 85% for LSA for p atients with RA. Discussion Several st udies were p erformed about the determination of a new marker for the diagnosis of rheumatoid arthritis. The p resent data confirms the p revious evidence that TSA and LSA are significantly higher in p atients with rheumatoid arthritis(RA).RA which is along –term disease that causes inflammation of joints and surrounding tissues. It can also affect other organs .M ost glycoproteins contain mannose ,galactose, fucose, sialic acid and in some instances glucose as the carbohydrate moiety . Sp iro, in 1959 established that liver is the major organ involved in the normal sy nthesis of glycoproteins [22].All the TSA and LSA were significantly elevated in the p lasma of p atients with(RA),T P from p atients of RA which showed minimal but st atist ically insignificant elevation . TSA and LSA may ,thus be able to differentiate an inflammatory arthritis from a dehydrative ,non-inflammatory arthritis .Our results are sup p orted with p reviously p ublished work [23]. Previous st udies were centered largely around the demonst ration of increased levels of carbohy drates of the carbohy drate-p rotein comp lex and carbohy drate – lip id comp lex in p lasma of RA. M ore over, elevated p lasma or serum levels of TSA and / or LSA were observed in many cases (23 - 25). There was a positive correlation of ESR with glycoproteins st udied, the best correlation being with hexosamine followed by sialic acid and other hexose in that order[26].The elevation of p lasma TSA and LSA in RA p atients is of note and we believe our data add to the literature showing changes in TSA and LSA st atus in RA. The mechanisms are unclear and we can only sp eculate as to the reason . In conclusion, the increase of p lasma TSA and LSA levels is associated p ositively with the p resence of inflammation and app ears t o be a consequence of the disease itself, and could be suggested as one of the newly discovered marker for RA. Re ference 1- Schauer R .and kelm S,etc (Biochemistry and role of Sialic acid .Rosenberg A eds. Biolojy of the sialic acid 1995,plenum publishin g Crop .New y ork01 2- Novak, J.; Tomana, M.;Shah, GR.; Brow n, R.and Mestecky, J. (2005), J. 901.-(10): 89784Dent. Res., 52-:4735 )1997(clin c hem. Clin b iochem,.SG ,rsherKi-Fang -3 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 4-Witczak,Z.J.and Nieforth.K. (1997) Carbohydrate in drugs Design, Marcel Dekker, New york, 5- Mahmood ,T.J. and Ahmed, S.A. (2008), JMCZ, Hawler Medicinal Univ. (in Press). :94030 clin c hem., )1984( et al,. K.A ,Bhargava-6 42-(2):13713, Ann clin lab sci,)1983( .K.M,and Sc hwartz .M.A,Dinstrian -7 9-(2):40455,,Cancer )1985(,et alJones JD and .M.K ,Erbil-8 5-(12):527042, ,ecR,Cancer )1982(et al .Y ,Hirshaut.and N ,odisKatop-9 6-(3):16323 J surg oncol,(1983)et al.HJ. ,Keller.;U ,Khanderia-10 11- Wivedi, C.D.; Dixit, M.et al, (1990) cellular and molecular life sciences 94-91:(1)46, :41 BMC plum med)2001( ,et al.I ,Gulba.and I ,Turgut-21 1335.-: 133125etal (2002), Diabetes, .J.A ,Reichelt .M,Srihana -13 9. –(6): 1 40Hygiene, (2006), and J. Dent..C .J ,Lux –14 15-Kulkarni, A.V.and Engineer, J.J. (1986)et al, inflammation in rheumatoid 93-(2):8932,ournal of postgraduate med.disorder,J 672-:6646J.Rheumatol.,)1979( .P ,.and GarbrilW.Denko,C-61 11-:60424Svenneehlom,L.(1957).Biochemica biophysica acta.-71 180.-:17130(1980)Res commun chem. Path phrmacol.,N.stock ccKatopodis8.1 5275-:527042al,Caner Res (1982) N;Hirshaut,etKatopodis-91 751:177J.Biol.chem.,)1949( Bardawill,et al,and .C.Gorna-20 21-Tietz, N .W. (1999)Text book o clinical c hemistry ,3 rd Ed.A.C.Burtis,E.R.Ashwood,W.B.s ounders;p 477-530 748-:742234 J.biol.c hem)1959(.Spiro,R,G-22 23- Alturfan A. Ata .and Uslu E. et al, 2007 The tohoku . J.of experimental 248-:241213 medicine, 495-:49450 J.clin.pathol.) 1997( et al, .S ,constable .andM ,rookC -42 9-(3):17410 ,Int.J.biol.Marker.)1995(Lopez,seaz,et al-52 2283-(11):227964, Cancer,)2006( M.d,et al. , Rudolf Voigtmann-62 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 F ig -4 -L e v e ls o f L S A f ro m p la s m a o f R A a n d c o n tr o l g ro u p 5 3 .1 3 8 8 .4 8 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 n o rm a l rh e u m a to id a rt hr it is LSA mg/dl F ig -3 -L e v e ls o f T S A f ro m p la s m a o f R A a n d c o n tr o l g ro u p 5 3 .1 3 8 8 .4 8 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 no rm a l rh e u m a to id a rt h ri ti s TSA mg/dl IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Fig-1-Percent of TSA, LSA and TP from pl asma of control gro up TSA 68% T P 9% LSA 23% Fig -2-P ercent o TSA, LSA an d TP from plasma of RA patients TSA 72% L SA 22% TP 6% Fi g-5 -Le vels o f TP from plas ma o f R A pa teints and c ontrol grou p 7. 35 7.39 7.33 7.34 7.35 7.36 7.37 7.38 7.39 7. 4 normal rh eumatoi art hriti s T P g /d l Series1 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 0 5 10 15 20 25 30 3 6 6.5 7 7.5 8 8.5 9 TP g/dl Fi g 6:-Correlation between TSA and LSA 0 20 40 60 80 100 120 140 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 L SA mg/dl T S A m g /d l 0 20 40 60 80 100 120 140 6 6.5 7 7.5 8 8.5 9 TP g/dl F ig 6 -c :-C o o re la tio n b e tw e e n L S A a n d T P L S A m g /d ll F ig -6 -b -c o rre la tio n b e tw e en T S A a n d T P T S A m g /d l IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Table (1): Data obtained revealing different statistical parameters Sensitivity selectivity P- value Confidence –interval Variance M ean+_SD mg/dl Range M g/dl Disease Parameter %10 %82.5 P<0.05 %95 200.34 88.48+_14.15 52.65- 122.33 Rheumatoid Art hritis N=40 TSA %95 57.48 53.31+_7.58 35.48- 69.41 Normal N=40 %12.5 %85 P<0.05 %95 5.08 26.37+_2.25 23.02- 30.85 Rheumatoid Art hritis N=40 LSA %95 2.11 18.46+_1.45 15.33- 21.96 Normal N=40 %22.5 %60 P<0.05 %95 0.11 7.39+_0.34 g/dl 7.01- 8.02 g/dl Rheumatoid Art hritis N=40 TP %95 0.11 7.35+_0.33 g/dl 7.04- 8.0 g/dl Normal N=40