IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Comparative Study of Fucose, Protein, Protein Bound Fucose and Protein Bound Hexose in Sera o f Hyperprolactinemia Human Female and Healthy Female Receivd in : 11 November 2008 Accepte d in : 30 June 2010 W.F. Al-Taie and M. A. Al-Iqabi Departme nt of Chemistry, College of Education, Ibn Al-Haitham, Baghdad Unive rsity Abstract This st udy was conducted in the sp ecialized center of endocrinology and diabetic (Al- Kindy hosp ital from June 2004 to Ap ril 2005). Sera of 80 women (include 40 diagnosed Hy p erprolactinemia (HPro) and 40 healthy women as control) were used to estimate some biochemical p arameters which include p rolactin (PRL),total fucose (TF), total p rotein (TP) and protein bound fucose (PBF), and p rotein bound hexose (PBHex), also TP TF , TP PBF and TP PBHex ratios. A significant elevation in TP and PBHex, in sera of HPro p atients comp ared to control was found while PBHex/TP ratio showed a slight non significant increase in sera of p atients comp ared to control. On the other hand a significant decrease in TF, TF/TP and PBF/TP in sera of HPro comp ared to control was found. Total fucose level (TF) in sera of HPro comp ared to control are (6.54  1.52), (10.66 4.00) mg/dL resp ectively. Prot ein bound fucose level (PBF) in sera of HPro comp ared to control are (3.88  1.25), (4.48 1.63) mg/dL resp ectively, while p rotein bound hexose (PBHex) level in sera of HPro comp ared to control are (131.111.43) (123.640.497) mg/dL resp ectively. The significant increase in PRL levels in sera of p atients was correlated with the significant reduced level of fucose in sera of patients comp ared to control. The low T F levels in patients could be due to the requirements of PRL variants to different ratios of monosaccharides including fucose and manose for glycosy lation of PRL which may alter the biological activity in different sp ecies and variety of mammalian. Key words: Fucose, Fucose Bound Protein , Hyperprolactine mia Introduction Prolactin (PRL) is a p olyp eptide hormone that is sy nthesized in and secreted from sp ecialized cells of the anterior p ituitary lobe the lactotrop hs [1]. The most relevant actions of p rolactin in the mammalian body are [2] Lactation, Luteal function, Female receptivity [3] and Parental behavior [4]. PRL-R are also p resent in a wide range of p erip heral organs like the p ituitary gland, heart, lung, thy mus, sp leen, liver, p ancreas, kidney , adrenal gland, uterus, skeletal muscle, and skin[5]. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 M arkedly raised blood PRL levels (>30ng/mL), might interfere with both endometrial p roliferation and follicle growt h and development and thereby reduce the likelihood of a successful pregnancy occurring [6]. Fucose (6-deoxy -L-galactose), a mono saccharide is p resent in low concentrations in normal circulation [7]. Fucose is found in a wide variety of natural substances from many different sources and occurs in abundance in glycoproteins and glycolipids in animals and humans [8]. Gly coconjugates are p resent in sy naptic junction areas where nerve cells meet, imp lying a role in sy naptic membrane involvement in nerve imp ulse transmissions. Human test es germ cells are rich in fucose and glucose glycoconjugates, which are altered during germ cell differentiation [9]. Gly cosy lated PRL has been found in the p ituitary glands of a wide variety of mammalian, amp hibian, and avian sp ecies. The degree of glycosy lation varies from 1 to 60% among sp ecies and may also vary between reproductive states within sp ecies [10]. The aim of the present st udy is to measure TF, PBF, PBHex, TP levels and verify the data obtained to each other by estimation the ratio of TF/TP, PBF/TP, PBHex/TP , also try to find correlations between(Prol. and TF), (Prol. and PBF), (Prol. and PBHex), (TF and PBF). S ampling (S ubjects) In a p lane tube (no anti coagulant),10 mL of venous blood was p laced, which was taken from the group s, left for (15 min) at room temperature, then centrifuged (at 2500 rp m for 10min) to get t he serum, which is stored at (-20 o C) unless used immediately . Col lection of blood The samples were collected from women p atients with HPro at the diagnosis time at (Sp ecialized center of endocrinology and diabetes, Al-Kindy Hosp ital). They were classified into two group s as follows: 1) Control group : includes (40) healthy women whose ages ranged between (15-43) y ears old, with no p revious diseases, which may interfere with the p arameters analyzed in this st udy . 2) Hy p erprolactinemia women group : includes (40) women whose ages ranged between (16-53) y ears old suffering from HPro. La boratory work. -An automated quantitative test was used on the VIDAS instruments, for the enzyme immunoassay determination of PRL in human serum or plasma (lithium heparinate) usin g enzy me linked fluorescent assay (ELFA). The assay p rinciple combines an enzy me immunoassay sandwich method with a final fluor escent detection (ELFA) [11,12]. -Total p rotein (TP) was determined accordin g to Biuret methods[13] which depends on the reaction of peptide bond of the p rotein with copp er ion (Cu ++ ) in alkaline medium to form colored products, whose absorbance is measured by (UV-Vis) at (540nm). -Total fucose (TF) was determined by using Dische and Shett les methods [14] in a direct reaction of concentrated sulp huric acid with serum comp onents. The reactants were combined with cyst eine, and the colored p roduct was measured at (390 and 430nm). The differences in absorbance were directly p rop ortional to -L-fucose content of the solutions. -Prot ein bound fucose (PBF) was determined according to Dische and Shett les methods [14] based on colored p roduct (chromophor) was formed by fucose in st rong acid medium, which IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 combined with color developer (Cy st ein hy drochloride). The colored p roduct with cyst eine measured at 390 nm, and 430nm resp ectively. -The orcinol reaction [15] is used to determine p rotein bound hexose (PBHex), by p recipitating the hexose moiety of p rotein-carbohy drate conjugate with ethanol (95%) at room temp erature, which measured at 520 nm. S tatistical anal ysis Data p resented were the means and st andard deviations; st udent-t -test was used to comp are the significance of the difference in the mean values of any two group s. (P0.05) was considered statist ically significant [16]. The overall p redictive values for the results in all st udied group s were p erformed according to p rogram of Office XP 2002. Results and Discussion The results of serum p rolactin in Hyp erp rolactinemia f emales and control are shown in the table (1). M any factors control, the secretion of PRL p hy siologically, PRL levels are controlled by the hyp othalamus [17]. Hy p erprolactinemia (HPro) has been recognized as a cause of infertility p roblems in men and women [18]. The three etiological forms of HPro are iatrogenic HPro associated with the use of certain medications (e.g. anti-depressants, tranquilizers), p rimary HPro associated with p ituitary tumors[19], and secondary HPro (hy p erthy roidism, renal insufficiency )[20]. The release of PRL is assessed at the single-cell level, the p att ern of PRL secretion of individual lactot rop hs shows sexual dimorp hism. In general, sl ightly more than half, the lactot rop hs of female rats secrete PRL in a continuous p attern[21]. Several invest igations have been concluded that, calcitonin-like p eptides are effective in inh ibiting PRL secretion when ad ministered in vivo [22] . From the table(2) there was a significant increase in TP level between HPro group and control with P value equals 0.003. A significant decrease in tot al fucose was found between HPro and control, also a significant decrease in TF/TP ratio. For HPro comp ared to control was found. The plasma levels of p roteins dep end on the balance between their sy nthesis and their degr adation[23]. Since PRL is a p rotein hormone of the anterior p ituitary gland and can be sy nthesized and secreted which is not restricted to the anterior p ituitary gland, but other organs and tissues in the body have this cap ability [24] .Some st imulators of p ituitary PRL secretion also affect hy p othalamic PRL p roduction, for examp le, ovarian st eroids modulate hy p othalamic sy nthesis and release of PRL[25,26]. Fucosy lated glycans have been amp licated in the p athogenesis of several human diseases[27]. High levels of fucose were reported in sera of hepato cellular carcinoma p atients and diabetes mellitus[28,29], and in leukemic p atients[30]. The reduction in TF for HPro p atients could be due to the high biological activity of the anterior p ituitary which required higher glycosy lation. Where the carbohydrate residues contain varying ratios of sialic acid, IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 fucose, manose and galactose which differ considerably between sp ecies, p hy siological and p athological and p athological st ates[31,32,10]. The disturbance in the level of PBF and PBHex in p atients could be due to the fucoligands in the cells of interest[10]. Gly cosy lation of the asp aragy l residues (Asn 35 , Asn 80 , Asn 108 ) of the extracellular domain of the PRL-R is crucial, although not absolute requirement for PRL-R activation. Although PRL- R is mainly a cell-surface receptor, deglycosy lated forms of PRL-R can be accumulated in the Golgi ap p aratus[33]. Nitric oxide activates N-acety lglucosamine transferase, which is resp onsible for glycosy lation of these intracellular receptors and p romotes migration of these newly glycosy lated recep tors t o the cell surface[34]. Correl ation relation The correlation coefficient (r) test is used to describe the association between the different st udied p arameters, P<0.01 was considered st atist ically significant. 1- Correlation relation between p rolactin PRL and tot al fucose TF Figures (1-A) and (1-B) showed a high significant p ositive correlation between PRL and TF marker with P value 0.0001 for hy p erprolactinemia (HPro) and control group s with correlation coefficient (r) values (0.775), (0.789) resp ectively. 2. Correlation relation between PRL and p rotein bounded fucose PBF. Figures (2-A) and (II-B) showed a high significant p ositive correlation between PRL and PBF marker with P value 0.0001 for HPro and control group s with correlation coefficient (r) value (0.923), (0.769) resp ectively. 3. Correlation relation between TF and PBF Figures (3-A) and (III-B) showed a high significant p ositive correlation between TF and PBF marker with P value 0.0001 for HPro and control group s with correlation coefficient (r) value (0.893), (0.862) resp ectively. Re ferences 1. Riddle, O.; Bates, R.W. and Dy kshorn, S.W. (1933) Am J p hy siol; 105: 191-216, 2. Bole-Fey sot , C.; Goffin, V.; Edery, M .; Binart, N. and Kelly, P.A. Endocr Rev; 19: 225- 268, (1998). 3. Dut t, A.; Kap litt, M .G.; Kowl, M . and Pfaff, D.W. Neuroenocrinology ; 59: 413-419, (1994). 4. Bridges, R.S. Acta paediatr; Sup p l 397: 33-39 (1994). 5. Nagano, M . and Kelly, P.A. (1994). J Biol Chem; 269: 13337-13345, 6. M erchenthalar, I.; Lennard, D.E.; Cianchetta, P.; M erchenthalar, A. and Bronst ein, D. (1995).Endocrinology 136: 2442-2450, 7. Srefani, M .R., Dunlap, J.A., and Yorek, M .A.: J. Cell: Phy siol; Nov, 153(2): 321-331, (1992). 8. Flower, H.M . (1981) Adv. Carbohy dr Chem. Biochem; 39: 279-345 9. M almi, R., Kallajoki, M ., and Suominer, J. (1987).Andrologia' M ay 19: 322-332 10. Sinha, Y.N. (1995) Endocr Rev, 16: 354-369 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 11. Hermin, L. M . (1976) "Prolactin Hormone Assays and their clinical Ap p lication" 4eme ed. Edite p ar Loraine, J. A., and Bell, E.T. Churchill Living st one, Edinburg PP. 293- 332, 12. Bardin, C.W. and Paulsen, C.A. (1981) "Textbook of endocrinology " 6eme Ed Williams, H.R. and Saunders, W.B. Philadelphia, 13. Oser, B.L. (1968) Hawk’s Phy siological Chemistry .14 th ed .M c. Graw-Hill, New York, PP 179-181, 14. Dische, Z., and Shett les, L.B. (1948) J. Biol. Chem; 176: 595-603, 15. Rimingto, C. (1990) J. Biochem, 34: 931 16. Bailey , N.I. (1974) "Statistical M ethods in Biology ", Ltd. Press, 17. Ben-Jonathan, N. (1985)Dop amine: a p rolactin –inhibiting hormone. Endocr Rev 6: 564-589 [ISI] [M edline]. 18. Djiane, J. and Kelly , P.A. (1984) Flammanon M edecine-Sciences-p resses del Universite de M ontreal, PP 141-146, 19. Allolo, B.; Hoep p ener, A. and Leohardt, U. (1987). Acta Endocrinological (Cop enh) 114: 475-482, 20. Franks, S. (1983) "Prolactin Hormones in Blood" 4, 3eme ed. Edite p ar Gray C.H. et James V.H.T. Academic p ress PP. 109-136, 21. Castano, J.P. and Frawley , L.S. (1995) Am J Phy siol Endocrinol M etab; 269: E 814- E 819 22. Fahim, A.; Rellori, V. and M .C. Cann, S.M . (1990) Neuroendocrinology ; 51: 688-693, 23. Zilva, P.M . and Philip, D.M . (2002) "Clinical chemist ry in diagnosis and treatment" 6 th ed. PP. 159-160, 165-233 24. Bern, H.A. and Nicoll, C.A. (1968) Rec Prog Horm Res; 24: 681-720 25. Devito, W.J.; Avakian, C.; Stone, S. and Ace, C.L. (1992) Endocrinology ; 131: 2154- 2160, 26. Devito, W.J.; Stone, S. and Vakian, C.A. (1997) Neuroendocrinology ; 54: 391-398 (1991). 27. Kim, Y.J. and Varki, A. (1997) Glycoconj. J. 14: 569-576, 28. Hassan, H.G.(2004) Ibn-Al-Haitham J. For p ure and Ap p l. Sci. 17(3):71-79, 29. AL-Haidary , S.M . (2004) "Evaluation of -L-fucose and related p arameters in different ty p es of diabetes mellitus" M Sc. Thesis; College of education, Baghdad University , 30. Leweza B.A. (2004) "Changes in Alp ha-L-Fucose and Related Parameters Levels in Leukemia" Ph.D. thesis College of M edicine University of Salahaddin-Erbil, 31. M arkoff, E., Sigel, M .B., Lacour, N., Seavey, B.K., Friesen, H.G., and Lewis, U.J. (1988) Endocrinology ; 123: 1303-1306, 32. Sinha Y.A., Dep aolo, L.V., Haro, L.S., Singh, R.N.P, Jacobsen B.P, Scott , K.E., and Lewis U.J.(1991) M olcell Endocrinol; 80: 203-213, 33. Buteau, H., Pezet, A., Ferrag, F., Perrot-Ap p lanat, M ., Kelly , P.A., and Edery , M . (1998) M ol Endocrinol; 12: 544-555, 34. Bolander, F.F. (1999) M ol cell Endocrionl; 149: 85-92, IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Table (1):Levels of PRL in sera of Hyperprolactine mia patients and control Group descrip tion No. Age PRL. M .I.U/mL meanSD P Control 40 15-43 14.20  5.75 Hy p erprolactinemia 40 16-53 45.74  36.88 < 0.05 Table (2):Levels of TP, TF and TF/TP ratio in sera of Hyperprolactine mia patients and control Group descrip tion No. TP g/dL meanSD P TF mg/dL meanSD P TF/TP mg/g meanSD P Control 40 6.44  0.20 10.66  4.00 1.65  0.61 Hy p erprolactinemia 40 6.71  0.54 0.003 6.54  1.52 0.05 0.98  0.22 < 0.05 Table (3):Levels of PBF, PBHex and PBF/TP, PBHex/TP ratio i n sera of Hyperprolactine mia patients and control Group description No. PB F mg/dL meanSD P PB F/TP mg/g meanSD P PB Hex mg/dL mean SD P PB Hex/TP mg/g meanSD P Control 40 4.48  1.63 0.70  0.25 123.64  0.49 19.22  0.59 Hyperprol actinemia 40 3.88  1.25 0.07 0.58  0.18 0.02 131.11  1.43 0.00 19.66  1.67 0.12 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Fig. (1-A): Correlation relati on between PRL an d TF in control group Fig. (1-B): Correlation relation between PRL and TF in Hyperprolactine mia patients group r ControL 0 2 4 6 8 10 12 14 16 18 0 5 10 15 20 25 PROLACTIN TF HYPERPROLACTINEMIA 0 2 4 6 8 10 12 14 0 50 100 150 200 250 PROLACTIN TF r=0.775 r=0.789 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Fig (II-A): Correlation relation between PRL and PBF in control group Fig .(2-A): Correlation relati on between PRL an d PBF in control group Fig. (2-B): Correlation relation between PRL and PB F in Hyperprolactine mia patients group ControL 0 1 2 3 4 5 6 7 8 0 5 10 15 20 25 PROLACT IN PBF HYPERPROLACTINEMIA 0 1 2 3 4 5 6 7 8 9 10 0 50 100 150 200 250 PROLACTIN PBF r=0.923 r=0.769 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (1) 2011 Fig (3-A): Correlation relati on between TF and PBF in control group Fig. (3-B): Correlation relation between TF and PBF in Hyperprolactine mia patients group ControL 0 1 2 3 4 5 6 7 8 0 2 4 6 8 10 12 14 16 18 TF PBF r= 0.893 2011) 1( 24المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة فوكوز المرتبط بالبروتین والسكریات ، البروتین، سة مقارنة لسكر الفوكوزدرا المرتبطة بالبروتین في مصول دم النساء المصابات بأرتفاع هرمون البروالكتین والنساء االصحاء 2008تشرین الثاني 11:استلم البحث في 2010حزیران 30: قبل البحث في اس العقابيمنتهى عب،وفاء الطائي جامعة بغداد ،الهیثم -كلیة التربیة ابن ،قسم الكیمیاء الخالصة الـى 2004ة مـن حزیـران مـدلاهذه الدراسة في المركز التخصصـي للغـدد الصـم والسـكري التـابع لمستشـفى الكنـدي یت إجر .2005غایة نیسان امـرأة مـن األصــحاء 40البـروالكتین و مـنهن مصـابات بارتفــاع فـي إفـراز هرمـون 40امـرأة 80مصـل دم لمعلقـد اسـت والبــروتین (TF)،والفوكـوز الكلــي ،(PRL)مجموعـة ســیطرة لتقـدیر بعــض الـدوال الكیموحیویــة التــي تضـم هرمــون مولـد الحلیــب نســـبة تكــذلك حســب PBHex)(والســكریات السداســیة المرتبطــة بـــالبروتین (PBF)والفوكــوز المــرتبط بـــالبروتین ،(TP)الكلــي (الكلـي إلـى البـروتین الكلـي الفوكـوز TP TF ( لكلـياونسـبة الفوكـوز المـرتبط بـالبروتین إلـى البـروتین ) TP PBF ونسـبة السـكریات ) (السداسیة المرتبطة بالبروتین إلى البروتین الكلي TP PBHex . ( . روتین الكلـــي والبـــروتین المـــرتبط بالســــكریات السداســـیة والكالســـیوم الكلــــيلقـــد وجـــدت زیـــادة معنویــــة فـــي مســـتوى البــــ بینما ،البروتینات الدهنیة العالیة الكثافة في مصول دم النساء المصابات بارتفاع في الهرمون المولد للحلیب مقارنة مع األصحاء لبــروتین الكلــي ارتفاعــًا غیــر معنــوي مقارنــة مــع أظهـر مســتوى الكولســترول ونســبة البــروتین المــرتبط بالســكریات السداســیة إلـى ا .مجموعة السیطرة و TFلقد وجد انخفاض معنوي في مستوى الـ TP TF و TP PBF في مصول دم النساء المریضات مقارنة مع مجموعة .قلة غیر معنویة في مصول دم المریضات مقارنة بمجموعة السیطرة) PBF(كما أظهرت مستویات ال،السیطرة رمـون المولـد للحلیـب مقارنـة مـع األصــحاء إن مسـتوى الفوكـوز الكلـي فـي مصـول دم المریضـات المصــابات بارتفـاع اله .مل على التوالي100/ملغم (10.664.00)و (6.541.52)هي مــل 100/ملغــم (4.481.63)و (3.881.25)إن مسـتوى البــروتین المـرتبط بــالفوكوز فــي مصـول المریضــات هـي فـي مصـل دم المریضـات المصـابات بارتفـاع هرمـون البـروالكتین (PBHex)على التوالي والبروتین المرتبط بالسكریات السداسـیة (131.111.43) و(123.640.49) مل على التوالي الزیادة المعنویة في مستوى البروالكتین في 100/غممل 2011) 1( 24المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة مقارنـة مـع أنفسـهن مع المسـتویات المنخفضـة معنویـًا للفوكـوز فـي مصـول المریضـات مصل دم المریضات تم ربطها .مجموعة السیطرة یعــزى االنخفــاض فــي مســتوى الفوكــوز الكلــي فــي مصــول دم المریضــات لمتطلبــات البــروالكتین بمختلـــف األوزان وقــد Glyالجزیئیـــة إلـــى اخــــتالف نســـب الســـكریات األحادیــــة والتـــي تشــــمل الفوكـــوز والمـــانوز لعملیــــة تســـّكر cosy lation هرمــــون .ف واألنواع المختلفة من اللبائنالبروالكتین الذي قد یغیر من الفعالیة البیولوجیة له في األصنا البروتین المرتبط مع الفوكوز، فرط هرمون البروالكتین، الفوكوز :الكلمات المفتاحیة