IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 Comparative Study of Fucose, Protein, Protein Bound Fucose and Protein Bound Hexose in Sera of Hyperprolactinemia Human Female and Healthy Female Receivd in : 11 November 2008 Accepted in : 30 June 2010 W.F. Al-Taie and M. A. Al-Iqabi Department of Chemistry, College of Education, Ibn Al-Haitham, Baghdad University Abstract This study was conducted in the specialized center of endocrinology and diabetic (Al- Kindy hospital from June 2004 to April 2005). Sera of 80 women (include 40 diagnosed Hyperprolactinemia (HPro) and 40 healthy women as control) were used to estimate some biochemical parameters which include prolactin (PRL),total fucose (TF), total protein (TP) and protein bound fucose (PBF), and protein bound hexose (PBHex), also TP TF , TP PBF and TP PBHex ratios. A significant elevation in TP and PBHex, in sera of HPro patients compared to control was found while PBHex/TP ratio showed a slight non significant increase in sera of patients compared to control. On the other hand a significant decrease in TF, TF/TP and PBF/TP in sera of HPro compared to control was found. Total fucose level (TF) in sera of HPro compared to control are (6.54  1.52), (10.66 4.00) mg/dL respectively. Protein bound fucose level (PBF) in sera of HPro compared to control are (3.88  1.25), (4.48 1.63) mg/dL respectively, while protein bound hexose (PBHex) level in sera of HPro compared to control are (131.111.43) (123.640.497) mg/dL respectively. The significant increase in PRL levels in sera of patients was correlated with the significant reduced level of fucose in sera of patients compared to control. The low TF levels in patients could be due to the requirements of PRL variants to different ratios of monosaccharides including fucose and manose for glycosylation of PRL which may alter the biological activity in different species and variety of mammalian. Key words: Fucose, Fucose Bound Protein , Hyperprolactinemia Introduction Prolactin (PRL) is a polypeptide hormone that is synthesized in and secreted from specialized cells of the anterior pituitary lobe the lactotrophs [1]. The most relevant actions of prolactin in the mammalian body are [2] Lactation, Luteal function, Female receptivity [3] and Parental behavior [4]. PRL-R are also present in a wide range of peripheral organs like the pituitary gland, heart, lung, thymus, sp leen, liver, pancreas, kidney, adrenal gland, uterus, skeletal muscle, and skin[5]. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 Markedly raised blood PRL levels (>30ng/mL), might interfere with both endometrial proliferation and follicle growth and development and thereby reduce the likelihood of a successful pregnancy occurring [6]. Fucose (6-deoxy-L-galactose), a mono saccharide is present 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]. Glycoconjugates are present in synaptic junction areas where nerve cells meet, implying a role in synaptic membrane involvement in nerve impulse transmissions. Human testes germ cells are rich in fucose and glucose glycoconjugates, which are altered during germ cell differentiation [9]. Glycosylated PRL has been found in the pituitary glands of a wide variety of mammalian, amphibian, and avian species. The degree of glycosylation varies from 1 to 60% among species and may also vary between reproductive states within species [10]. The aim of the present study 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). Sampling (S ubjects) In a plane tube (no anti coagulant),10 mL of venous blood was placed, which was taken from the groups, left for (15 min) at room temperature, then centrifuged (at 2500 rpm for 10min) to get the serum, which is stored at (-20 oC) unless used immediately . Collection of blood The samples were collected from women patients with HPro at the diagnosis time at (Specialized center of endocrinology and diabetes, Al-Kindy Hospital). They were classified into two groups as follows: 1) Control group: includes (40) healthy women whose ages ranged between (15-43) years old, with no previous diseases, which may interfere with the parameters analyzed in this study. 2) Hyperprolactinemia women group: includes (40) women whose ages ranged between (16-53) years old suffering from HPro. Laboratory 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) using enzyme linked fluorescent assay (ELFA). The assay principle combines an enzyme immunoassay sandwich method with a final fluorescent detection (ELFA) [11,12]. -Total protein (TP) was determined according to Biuret methods[13] which depends on the reaction of peptide bond of the protein with copper 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 Shettles methods [14] in a direct reaction of concentrated sulphuric acid with serum components. The reactants were combined with cysteine, and the colored product was measured at (390 and 430nm). The differences in absorbance were directly proportional to -L-fucose content of the solutions. -Protein bound fucose (PBF) was determined according to Dische and Shettles methods [14] based on colored product (chromophor) was formed by fucose in strong acid medium, which IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 combined with color developer (Cystein hydrochloride). The colored product with cysteine measured at 390 nm, and 430nm respectively. -The orcinol reaction [15] is used to determine protein bound hexose (PBHex), by precipitating the hexose moiety of protein-carbohydrate conjugate with ethanol (95%) at room temperature, which measured at 520 nm. Statistical analysis Data presented were the means and standard deviations; student-t-test was used to compare the significance of the difference in the mean values of any two groups. (P0.05) was considered statistically significant [16]. The overall predictive values for the results in all studied groups were performed according to program of Office XP 2002. Results and Discussion The results of serum prolactin in Hyperprolactinemia females and control are shown in the table (1). Many factors control, the secretion of PRL physiologically, PRL levels are controlled by the hypothalamus [17]. Hyperprolactinemia (HPro) has been recognized as a cause of infertility problems 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), primary HPro associated with pituitary tumors[19], and secondary HPro (hyperthyroidism, renal insufficiency)[20]. The release of PRL is assessed at the single-cell level, the pattern of PRL secretion of individual lactotrophs shows sexual dimorphism. In general, slightly more than half, the lactotrophs of female rats secrete PRL in a continuous pattern[21]. Several investigations have been concluded that, calcitonin-like peptides are effective in inhibiting PRL secretion when administered 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 total fucose was found between HPro and control, also a significant decrease in TF/TP ratio. For HPro compared to control was found. The plasma levels of proteins depend on the balance between their synthesis and their degradation[23]. Since PRL is a protein hormone of the anterior pituitary gland and can be synthesized and secreted which is not restricted to the anterior pituitary gland, but other organs and tissues in the body have this capability[24] .Some stimulators of pituitary PRL secretion also affect hypothalamic PRL production, for example, ovarian steroids modulate hypothalamic synthesis and release of PRL[25,26]. Fucosylated glycans have been amplicated in the pathogenesis of several human diseases[27]. High levels of fucose were reported in sera of hepato cellular carcinoma patients and diabetes mellitus[28,29], and in leukemic patients[30]. The reduction in TF for HPro patients could be due to the high biological activity of the anterior pituitary which required higher glycosylation. Where the carbohydrate residues contain varying ratios of sialic acid, IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 fucose, manose and galactose which differ considerably between species, physiological and pathological and pathological states[31,32,10]. The disturbance in the level of PBF and PBHex in patients could be due to the fucoligands in the cells of interest[10]. Glycosylation of the asparagyl 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, deglycosylated forms of PRL-R can be accumulated in the Golgi apparatus[33]. Nitric oxide activates N-acetylglucosamine transferase, which is responsible for glycosylation of these intracellular receptors and promotes migration of these newly glycosylated receptors to the cell surface[34]. Correlation relation The correlation coefficient (r) test is used to describe the association between the different studied parameters, P<0.01 was considered statistically significant. 1- Correlation relation between prolactin PRL and total fucose TF Figures (1-A) and (1-B) showed a high significant positive correlation between PRL and TF marker with P value 0.0001 for hyperprolactinemia (HPro) and control groups with correlation coefficient (r) values (0.775), (0.789) respectively. 2. Correlation relation between PRL and protein bounded fucose PBF. Figures (2-A) and (II-B) showed a high significant positive correlation between PRL and PBF marker with P value 0.0001 for HPro and control groups with correlation coefficient (r) value (0.923), (0.769) respectively. 3. Correlation relation between TF and PBF Figures (3-A) and (III-B) showed a high significant positive correlation between TF and PBF marker with P value 0.0001 for HPro and control groups with correlation coefficient (r) value (0.893), (0.862) respectively. References 1. Riddle, O.; Bates, R.W. and Dykshorn, S.W. (1933) Am J physiol; 105: 191-216, 2. Bole-Feysot, C.; Goffin, V.; Edery, M.; Binart, N. and Kelly, P.A. Endocr Rev; 19: 225- 268, (1998). 3. Dutt, A.; Kaplitt, M.G.; Kowl, M. and Pfaff, D.W. Neuroenocrinology; 59: 413-419, (1994). 4. Bridges, R.S. Acta paediatr; Suppl 397: 33-39 (1994). 5. Nagano, M. and Kelly, P.A. (1994). J Biol Chem; 269: 13337-13345, 6. Merchenthalar, I.; Lennard, D.E.; Cianchetta, P.; Merchenthalar, A. and Bronstein, D. (1995).Endocrinology 136: 2442-2450, 7. Srefani, M.R., Dunlap, J.A., and Yorek, M.A.: J. Cell: Physiol; Nov, 153(2): 321-331, (1992). 8. Flower, H.M . (1981) Adv. Carbohydr Chem. Biochem; 39: 279-345 9. Malmi, R., Kallajoki, M., and Suominer, J. (1987).Andrologia' May 19: 322-332 10. Sinha, Y.N. (1995) Endocr Rev, 16: 354-369 IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 11. Hermin, L. M. (1976) "Prolactin Hormone Assays and their clinical Application" 4eme ed. Edite par Loraine, J. A., and Bell, E.T. Churchill Living stone, 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 Physiological Chemistry.14 th ed .Mc. Graw-Hill, New York, PP 179-181, 14. Dische, Z., and Shettles, L.B. (1948) J. Biol. Chem; 176: 595-603, 15. Rimingto, C. (1990) J. Biochem, 34: 931 16. Bailey, N.I. (1974) "Statistical Methods in Biology", Ltd. Press, 17. Ben-Jonathan, N. (1985)Dopamine: a prolactin –inhibiting hormone. Endocr Rev 6: 564-589 [ISI] [Medline]. 18. Djiane, J. and Kelly, P.A. (1984) Flammanon Medecine-Sciences-presses del Universite de Montreal, PP 141-146, 19. Allolo, B.; Hoeppener, A. and Leohardt, U. (1987). Acta Endocrinological (Copenh) 114: 475-482, 20. Franks, S. (1983) "Prolactin Hormones in Blood" 4, 3eme ed. Edite par Gray C.H. et James V.H.T. Academic press PP. 109-136, 21. Castano, J.P. and Frawley, L.S. (1995) Am J Physiol Endocrinol Metab; 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 chemistry 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 pure and Appl. Sci. 17(3):71-79, 29. AL-Haidary, S.M. (2004) "Evaluation of -L-fucose and related parameters in different types of diabetes mellitus" M Sc. Thesis; College of education, Baghdad University , 30. Leweza B.A. (2004) "Changes in Alpha-L-Fucose and Related Parameters Levels in Leukemia" Ph.D. thesis College of Medicine University of Salahaddin-Erbil, 31. Markoff, 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., Depaolo, 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-Applanat, M., Kelly, P.A., and Edery, M. (1998) Mol Endocrinol; 12: 544-555, 34. Bolander, F.F. (1999) M ol cell Endocrionl; 149: 85-92, IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 Table (1):Levels of PRL in sera of Hyperprolactinemia patients and control Group description No. Age PRL. M.I.U/mL meanSD P Control 40 15-43 14.20  5.75 Hyperprolactinemia 40 16-53 45.74  36.88 < 0.05 Table (2):Levels of TP, TF and TF/TP ratio in sera of Hyperprolactinemia patients and control Group description 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 Hyperprolactinemia 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 in sera of Hyperprolactinemia patients and control Group description No. PBF mg/dL meanSD P PBF/TP mg/g meanSD P PBHex mg/dL mean SD P PBHex/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. SCI. VOL.24 (1) 2011 Fig. (1-A): Correlation relation between PRL and TF in control group Fig. (1-B): Correlation relation between PRL and TF in Hyperprolactinemia 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. SCI. VOL.24 (1) 2011 Fig (II-A): Correlation relation between PRL and PBF in control group Fig .(2-A): Correlation relation between PRL and PBF in control group Fig. (2-B): Correlation relation between PRL and PBF in Hyperprolactinemia patients group ControL 0 1 2 3 4 5 6 7 8 0 5 10 15 20 25 PROLACTIN 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. SCI. VOL.24 (1) 2011 Fig (3-A): Correlation relation between TF and PBF in control group Fig. (3-B): Correlation relation between TF and PBF in Hyperprolactinemia 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 ـكریات السداســیة المرتبطــة بـــالبروتین (PBF)والفوكــوز المــرتبط بـــالبروتین ،(TP)الكلــي نســـبة تكــذلك حســب PBHex)(والسـ (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 . ( ة فـــي مســـتوى البــــ ـكریات السداســـیة والكالســـیوم الكلــــيلقـــد وجـــدت زیـــادة معنویــــ . روتین الكلـــي والبـــروتین المـــرتبط بالســـ بینما ،البروتینات الدهنیة العالیة الكثافة في مصول دم النساء المصابات بارتفاع في الهرمون المولد للحلیب مقارنة مع األصحاء ـكریات السداســیة إلـى ا لبــروتین الكلــي ارتفاعــًا غیــر معنــوي مقارنــة مــع أظهـر مســتوى الكولســترول ونســبة البــروتین المــرتبط بالسـ .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 ة مـع أنفسـهن مع المسـتویات المنخفضـة معنویـًا للفوكـوز فـي مصـول المریضـات مصل دم المریضات تم ربطها مقارنـ .مجموعة السیطرة ـات البــروالكتین بمختلـــف األوزان وقــد یعــزى االنخفــاض فــي مســتوى الفوكــوز الكلــي فــي مصــول دم المریضــات لمتطلبـ ــّكر ـكریات األحادیــــة والتـــي تشــــمل الفوكـــوز والمـــانوز لعملیــــة تسـ ة إلـــى اخــــتالف نســـب الســ هرمــــون Glycosylationالجزیئیـــ .ف واألنواع المختلفة من اللبائنالبروالكتین الذي قد یغیر من الفعالیة البیولوجیة له في األصنا البروتین المرتبط مع الفوكوز، فرط هرمون البروالكتین، الفوكوز :الكلمات المفتاحیة