The effect of acetaldehyde-glycosaminoglycan mixtures upon Factor IXa and Factor IX-Deficient Plasma

Alcohol 39 (2006) 97e104

The effect of acetaldehyde-glycosaminoglycan mixtures upon Factor IXa and Factor IX-Deficient Plasma Arthur S. Brecher*, Amie Rebecca Moon, Kelly D. Gray Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403, USA Received 29 March 2006; received in revised form 27 July 2006; accepted 22 August 2006

Abstract Earlier studies have shown that acetaldehyde, the primary intermediate in the biological degradation of ethanol, interacts with enzymes and zymogens of the common coagulation pathway, prolonging prothrombin time and activated partial thromboplastin time (APTT), and that acetaldehyde-glycosaminoglycans (GAGs) mixtures synergistically prolong clotting times (Brecher, A. S. (2005). In Comprehensive Handbook of Alcohol Related Pathology. Vol. 3(93), pp. 1223e1244). In this study, the effect of acetaldehyde and GAGs upon Factor IXa, an intrinsic pathway enzyme, has been investigated. Individually, acetaldehyde, heparins of various molecular weights, dermatan sulfate, and chondroitin sulfates A and C affect Factor IXa, prolonging clotting time as measured by APTT. Pre-incubation of Factor IXa with a mixture of 22.3 mM acetaldehyde and heparin17k, heparin6k, dermatan sulfate, or chondroitin sulfate A additively prolongs clotting times, reflecting individual, unrelated molecular mechanistic effects. In contrast, a synergistic effect is observed at the 44.7 mM acetaldehyde level with heparin17k, heparin3k, chondroitin sulfates A and C, and dermatan sulfate, suggesting that acetaldehyde may cross-link with the enzyme and the GAGs, forming tertiary complexes, further influencing coagulopathy. These observations upon Factor IXa present a deeper dimension to the anticoagulation effect of alcohol on the coagulation cascade. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Factor IXa; Blood coagulation; APTT; Acetaldehyde; Glycosaminoglycans; Alcoholism

1. Introduction A prolonged blood coagulation time is frequently observed in the alcoholic population (Podolsky & Isselbacher, 1994). Early on, this has been explained on the basis of a decreased biosynthesis of coagulation factors, generally as a result of poor nutrition whereby an insufficiency of vitamin K has been proposed (Crabb & Lumeng, 1989; Podolsky & Isselbacher, 1991). More recently, it has been additionally suggested, on the basis of laboratory experiments, that acetaldehyde (AcH), the primary metabolite/intermediate in ethanol metabolism, is capable of reacting with coagulation factors in a covalent manner, thereby inactivating them and prolonging clotting time (Basista et al., 1994; reviewed in Brecher, 2005). It had been reported that AcH prolonged the prothrombin time (PT) of commercial human plasma. In studies on the effect of AcH on components of the blood clotting system, it was noted that AcH reduced the functionality of thrombin, fibrinogen,

* Corresponding author. Tel.: þ1-419-372-2044; fax: þ1-419-3729809. E-mail address: [email protected] (A.S. Brecher). 0741-8329/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2006.08.005

thromboplastin, and Factor Xa, and furthermore decreased the extent of activation of prothrombin and Factor X (Basista et al., 1994; Brecher et al., 1996a, 1996b). AcH also reduced the capacity of antithrombin III (ATIII) to serve as an anti-coagulant (Brecher et al., 1998). In this latter behavior, AcH exerted a pro-coagulant effect. However, ATIII also exhibited an additional anti-coagulant effect upon interaction with heparin (Brecher et al., 1999). Whereas AcH and heparin each prolonged coagulation time, a mixture of the two compounds exhibited a synergistic effect in a PT assay. Further investigations with a PT assay revealed that synergism was observed upon pre-mixing AcH with chondroitin sulfates (CSs) A and C, as well as dermatan sulfate (Brecher and Adamu, 2001). When individual factors such as prothrombin and Factor Xa (FXa) were subjected to AcH-glycosaminoglycan mixtures, synergism was still noted, utilizing the activated partial thromboplastin time (APTT) assay (Brecher and Adamu, 2005; Brecher and Hommema, 2002). Additionally, effects on thrombin by AcH-heparin mixtures reflected synergism (Brecher and Fu, 1999). Whereas thrombin, prothrombin, and FXa are components of the common coagulation pathway, there is no

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information regarding effects on components of the intrinsic blood clotting system. Accordingly, this communication addresses the influence of mixtures of AcH at 22.3 and 44.7 mM concentrations with heparin17k, heparin6k, heparin3k, dermatan sulfate, and CSs A and C, as well as glycosaminoglycans (GAGs) alone on the interaction between Factor IXa (FIXa) and Factor IX-Deficient Plasma (FIXDP).

2. Materials and methods Acetaldehyde (11007-8, lot #JU12912AQ) and aluminum oxide, activated, basic, Brockman I (Cat#19, 944-3, Lot# 07911DY) were obtained from Aldrich Chemical Company, Inc., Milwaukee, WI, USA. Acetaldehyde was prepared by bubbling nitrogen gas through the sample, and then passing the sample through an A12O3 column to remove any oxidation products. The filtered acetaldehyde was then stored in 1 ml aliquots at 20 C. The concentration of this acetaldehyde was approximately 17.88 M. APTT reagent (B4219-2, lot#FSL-135A, FSL-163) and 0.02 M CaCl2 (CC-441B) were obtained from Baxter-Dade Diagnostics of P.R. Inc., Aguada, PR, USA. Additional APTT reagent (B4219-2, lot#SA-173) was obtained from Baxter Healthcare Corporation, Dade Division, Miami, FL, USA. A Fibrometer Precision Coagulation Timer equipped with a 0.3 ml probe arm and Thermal Prep Block Precision Heating Unit (Model #117) were purchased from BBL Division of Becton, Dickinson, and Company, Cockeysville, MD, USA. Fibrin Cups (02658101, lot#715301) were obtained from Elkay Sherwood Service AG, Tyco Healthcare Group LP, Mansfield, MA, USA. NaCl (BP358-1, lot#004726) was purchased from Fisher Scientific, Fair Lawn, NJ, USA. FIXa (HCIXa-0050, lot#N1201 and P0816) 12.2 mg/ml (12.2 mg/ml) and 9.3 mg/ml (9.3 mg/ ml), respectively, in a 50% (vol/vol) glycerol/dH2O solution, was purchased from Haematologic Technologies Inc. (HTI), Essex Junction, VT, USA. Imidazole (catalog #8862) was obtained from Matheson, Coleman, and Bell, Manufacturing Chemists, Norwood, OH USA. Bovine Albumin (A-3350, lot#93H0294) (Cohn Fraction V), Chondroitin Sulfate A (C-8529, lot#55H0306) (70%) isolated from Bovine Trachea, Chondroitin Sulfate B (C-3788, lot#94H0656) (90%) sodium salt (dermatan sulfate) isolated from porcine intestinal mucosa, Chondroitin Sulfate C (C-4384, lot#107H1029) (90%) isolated from shark cartilage, Heparin17k 190 U/mg (H-3393, lot#127H1161) isolated from porcine intestinal mucosa, Heparin6k (H-5284, lot#89H12121) from porcine intestinal mucosa, Heparin3k (H-3400, lot#98H1149) from porcine intestinal mucosa, and Trizma Base (T-1503, lot#109H5401) were purchased from Sigma Chemical Company, St. Louis, MO, USA. Factor IX-Deficient Plasma (F9D-1, lot#079H6020, 110K6046, and 041K6008) was obtained from Sigma Diagnostics, St. Louis, MO, USA.

2.1. Preparation of FIXa Three microliters of FIXa stock solution were diluted in 50% (vol/vol) glycerol/dH2O to yield a 1 mg/ml solution of FIXa which was stored at 20 C until use. A 0.1 mg/ml solution of FIXa was prepared fresh for each experiment by diluting 10 ml of the 1 mg/ml FIXa with 990 ml of a 50% (vol/vol) glycerol/dH2O solution. A working solution of 0.0045 mg/ml (4.5 ng/ml) FIXa was prepared by diluting 45 ml of the 0.1 mg/ml FIXa solution with 955 ml of 0.1 M NaCl, 0.05 M Tris/1 mg/ml BSA pH 7.3 buffer. Twenty microliters, (0.09 mg), of this solution was used for each assay. The solution was kept on ice throughout the experiment unless otherwise noted. 2.2. Effect of AcH on FIXa and FIXDP [FIXa þ ISB]: The control mixture consisted of 20 ml (0.09 mg) of cold FIXa diluted with 20 ml of cold ISB. Subsequently, 20 ml of the diluted mixture was added to 0.1 ml of cold FIXDP, followed by 90 ml of APTT reagent. The FIXa-FIXDP-APTT mixture was incubated at 37 C for 5 min. Immediately following the 5-min incubation, 90 ml CaCl2 at 37 C was added to the reaction mixture and the APTT was determined. [FIXa þ AcH]: A 20 ml (0.09 mg) aliquot of cold FIXa was incubated with 20 ml of cold AcH (437 mM) for 15 min on ice. The final concentration of AcH in the FIXa-AcH mixture was 223.5 mM. After 15 min, 20 ml of the cold FIXa-AcH experimental mixture was added to 0.1 ml of cold FIXDP, followed by 90 ml of cold APTT reagent, and the resulting mixture was incubated at 37 C for 5 min. The concentration of AcH in the FIXa-AcH-FIXDP mixture was 37.25 mM. Immediately following the 5-min incubation, 90 ml CaCl2 at 37 C was added to the FIXaAcH-FIXDP-APTT mixture and the APTT was determined. [(FIXDP þ AcH) þ FIXa]: A 0.1 ml aliquot of cold FIXDP was incubated with 10 ml of 410 mM cold AcH for 15 min on ice. The final concentration of AcH in the FIXDP-AcH reaction mixture was 37.25 mM. After 15 min, 10 ml (0.045 mg) of cold FIXa was added to the FIXDP-AcH mixture, followed by 90 ml of cold APTT reagent, and the FIXDP-AcH-FIXa-APTT mixture was incubated for 5 min at 37 C prior to assay as described above. 2.3. Effect of AcH concentration on FIXa and GAGs [FIXa þ ISB]: The primary control mixture consisted of 20 ml (0.09 mg) of cold FIXa incubated with 20 ml of cold ISB for 30 min at room temperature. After 27 min, 0.1 ml of cold FIXDP and 90 ml of cold APTT reagent were added to a fibrin cup and incubated for 3 min at 37 C. At 30 min, 20 ml of the control mixture was added to the FIXDP-APTT mixture, followed by 90 ml of CaCl2 at 37 C and the APTT was determined. [FIXa þ GAG]: A 20ml (0.09 mg) aliquot of cold FIXa was incubated with 10 ml of the appropriate cold GAG

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solution for 30 min at room temperature. Final quantities of GAG’s in the second control mixture were as follows: Heparin17k 5 0.05 mg (0.01 U), Heparin6k 5 0.1 mg, Heparin3k 5 0.5 mg, chondroitin sulphate B (CSB) 5 1.0 mg, chondroitin sulfate A (CSA) 5 25 mg, and chondroitin sulfate C (CSC) 5 10 mg. After 30 min, 10 ml of cold ISB was added to the FIXa-GAG control mixture to adjust the volume to a total of 40 ml. Subsequently, 20 ml of this solution was added to the FIXDP-APTT mixture for assay as described above. [FIXa þ AcH]: A 20 ml (0.09 mg) aliquot of cold FIXa was incubated with 10 ml of cold AcH (134 mM or 66.9 mM) for 30 min at room temperature. The final concentration of AcH in the third control mixture was 44.7 mM or 22.3 mM. After 30 min, 10 ml of cold ISB was added to the FIXa-AcH control mixture to adjust the volume to a total of 40 ml. Subsequently, 20 ml of this control mixture was taken and added to the FIXDP-APTT mixture for assay as described above. [(FIXa þ GAG) þ AcH]: A 20 ml (0.09 mg) aliquot of cold FIXa was incubated with 10 ml of the appropriate cold GAG solution for 30 min at room temperature. The final quantities of GAG’s were as follows: Heparin17k 5 0.05 mg (0.01 U), Heparin6k 5 0.1 mg, Heparin3k 5 0.5 mg, CSB 5 1.0 mg, CSA 5 25 mg, and CSC 5 10 mg. After 30 min, 10 ml of cold AcH (178 mM or 89.4 mM) was added to the FIXa-GAG reaction mixture and this mixture was incubated for an additional 30 min at room temperature. The final concentration of AcH in the FIXa-GAG-AcH experimental mixture was 44.7 mM or 22.3 mM. At 60 min, 20 ml of the FIXa-GAG-AcH mixture was added to the FIXDP-APTT mixture for assay as described above. [(FIXa þ AcH) þ GAG]: A 20 ml (0.09 mg) aliquot of cold FIXa was incubated with 10ml of cold AcH (134 mM or 66.9 mM) for 30 min at room temperature. The final concentration of AcH in the FIXa-AcH mixture was 44.7 mM or 22.3 mM. After 30 min, 10 ml of the appropriate cold GAG solution was added to the FIXa-AcH reaction mixture and the resulting mixture was incubated for an additional 30 min at room temperature. Final quantitites of GAG’s in the mixture were as follows: Heparin17k 5 0.05 mg (0.01 U), Heparin6k 5 0.1 mg, Heparin3k 5 0.5 mg, CSB 5 1.0 mg, CSA 5 25 mg, and CSC 5 10 mg. At 60 min, 20 ml of the experimental mixture was added to the FIXDP-APTT mixture for assay as described. [(GAG þ AcH) þ FIXa]: 10 ml of the appropriate cold GAG solution was incubated with 10 ml of cold AcH (178.8 mM or 89.4 mM) for 30 min at room temperature. The final quantity of GAG was as follows: Heparin17k 5 0.5 mg (0.01 U), Heparin6k 5 0.1 mg, Heparin3k 5 0.5 mg, CSB 5 1.0 mg, CSA 5 25 mg, CSC 5 10 mg. After 30 min, 20 ml (0.09 mg) of cold FIXa was added to the GAG-AcH mixture and the resulting solution was incubated for an additional 30 min at room temperature. The final concentration of AcH in the GAG-AcH-FIXa experimental mixture was 44.7 mM or 22.3 mM. At 60 min, 20 ml of the

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experimental reaction mixture was added to the FIXDPAPTT mixture for assay as described. 2.3.1. Effect of GAGs upon FIXDP To 100 ml FIXDP in a fibrometer cup were added 10 ml of ISB or 10 ml of the appropriate GAG, 10 ml FIXa (4.5 ng), and 90 ml APTT reagent. After standing for 5 min at 37 C, 90 ml of CaCl2 were added thereto and the clotting time was determined. 2.4. Statistical analysis The data were analyzed according to Student’s t-test. Values of P !.05 were considered statistically significant.

3. Results The effects of GAGs upon FIXa and FIXDP were studied (data not shown). H17k from 0.001 Ue0.1 U prolonged clotting time upon pre-incubation with FIXa to a greater extent than upon pre-incubation with FIXDP. H6k (0.05e0.5 mg) behaved similarly. However, H3k (0.5e2.5 mg) prolonged clotting time more effectively upon pre-incubation with FIXDP as compared to FIXa. The CSs each prolonged clotting times in uniquely different fashions. Dermatan sulphate (DS) (CSB) (1.0e10 mg) prolonged clotting time to a greater extent upon pre-incubation with FIXa as compared to FIXDP. CSC (10e50 mg) affected FIXa and FIXDP equally in prolonging APTT. CSA (10e50 mg) affected FIXa and FIXDP equally at 25 and 50 mg. At the 100 mg level, it prolonged the APTT to a greater extent upon pre-incubation with FIXDP. The effect of 37.3 mM AcH upon FIXa and FIXDP is given in Fig. 1. Whereas a 42.1 s increase in APTT was observed when the AcH was added to the FIXa, only a 1.6 s increase was noted upon addition of AcH to FIXDP, suggesting that the AcH has reacted with a wide variety of proteins in the FIXDP that are not associated with coagulation. The effect of AcH concentration upon FIXa was examined over a concentration range from 22.3 to 223.5 mM (Fig. 2), indicating progressively greater prolongation effects with greater acetaldehyde concentration. Statistically significant increases in APTT were observed at all pre-incubation concentrations of AcH with FIXa. The remaining experiments were performed with different FIXDP preparations than the initial experiments. Fig. 3 reflects the effect of mixtures of 0.05 mg H17k (0.01 U) with 44.7 or 22.3 mM AcH upon FIXa under three different pre-incubation conditions: [(FIXa þ H) þ AcH], [(FIXa þ AcH) þ H], and [(H þ AcH) þ FIXa]. At the 23 mM AcH level, essentially additive effects were observed with all pre-incubation conditions. At the 44.7 mM AcH level, a synergistic effect was observed at all pre-incubation conditions. The greatest synergism was observed, however, when [(FIXa þ AcH) þ H] mixtures were tested.

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Fig. 1. Effect of 37.25 mM acetaldehyde (AcH) on Factor IXa (FIXa) and Factor IX-deficient plasma (FIXDP) (Error bars represent standard error of the mean).

When H17k was replaced by 0.1 mg H6k in a similar pattern for pre-incubations, only additive effects of H6k and AcH or effects approaching the additive were observed (Fig. 4). The greatest additive observations were noted with preFig. 3. Effect of acetaldehyde (AcH) on Factor IXa (FIXa) and 0.05 mg (0.01 U) Heparin17k (Error bars represent standard error of the mean).

mixtures of [(FIXa þ AcH) þ H6k]. Mixtures of 0.5 mg H3k with both levels of AcH were by-and-large synergistic in prolonging APTT (Fig. 5). CSB (DS) (1 mg) in mixtures with 44 mM AcH affected a synergistic effect with FIXa, whereas 22.3 mM AcH exerted an additive effect with the GAG (Fig. 6). When 25 mg CSA were mixed with 44.7 mM AcH and FIXa, a synergistic effect was observed only with [(FIXa þ AcH) þ 25 mg CSA] pre-mixtures. All others at 44.7 mM as well as 22.3 mM exhibited additive effects (Fig. 7). The data in Fig. 8 illustrate the synergistic effect of 10 mg CSC with 44.7 mM AcH at all pre-mixtures, and an additional synergism at 22.3 mM AcH with a [(CSC þ AcH) þ FIXa] pre-mixture. All remaining premixtures showed additive effects of the GAGs with AcH, suggesting that each reagent reflects its own individual molecular effect in prolonging APTT.

4. Discussion Fig. 2. Effect of acetaldehyde (AcH) concentration on Factor IXa (FIXa) (Error bars represent standard error of the mean).

This laboratory has earlier reported effects of different GAGs upon coagulation. In a PT assay, H17k and DS

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Fig. 4. Effect of acetaldehyde (AcH) on Factor IXa (FIXa) and 0.1 mg Heparin6k (Error bars represent standard error of the mean).

prolonged prothrombin (PT) time, while CSA and CSC had no effect on PT (Brecher and Adamu, 2001). H17k also prolonged clotting times of thrombin in a thrombin-FIIDP assay (Brecher and Fu, 1999). The potential clotting time of prothrombin, as assayed by APTT, was also extended to a greater extent upon pre-incubation of H17k, 6k, 3k, CSA, DS, (CSB) with prothrombin as compared to FIIDP (Brecher and Adamu, 2005), suggesting a direct interaction effect for all the GAGs with prothrombin. A similar pattern was seen upon pre-incubation of the GAGs with FXa, as compared to pre-incubation with Factor X-Deficient Plasma (FXDP) (Brecher and Hommema, 2002). However, investigations on the effect of GAGs on enzymes associated exclusively with the intrinsic blood clotting system has been studied less extensively (Rosenberg, 1985; Zhao et al., 1998), and particularly not in association with AcH. This is of particular interest since low molecular weight heparins (LMWHs) are in clinical use. The current investigation was initiated in order to examine and to compare the influence of AcH and GAGs upon intrinsic factors with those of the common coagulation pathway. It was noted, by-and-large, that similar patterns of data were obtained with FIXa and FIXDP as compared with FXa and FXDP (Brecher and Hommema, 2002).

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Fig. 5. Effect of acetaldehyde (AcH) on Factor IXa (FIXa) with 0.5 mg Heparin3k (Error bars represent standard error of the mean).

Two levels of AcH were taken for exploration, 22.3 and 44.7 mM, in conjunction with levels of GAGs that gave modest, but definitive prolongations of clotting times by an APTT-type assay. The GAGs employed were heparin17k, heparin6k, and heparin3k, CSA and CSC, and dermatan sulfate. Experiments were designed to compare prolongation times (APTT) when pre-mixtures were executed in three ways: [(FIXa þ GAG) þ AcH], [(FIXa þ AcH) þ GAG], and [(GAG þ AcH) þ FIXa] for each of the AcH concentrations. In all cases, it was observed that [(FIXa þ AcH) þ GAG] mixtures gave the highest synergistic effect when heparin17k, heparin3k, dermatan sulfate, CSA, and CSC were utilized with 44.7 mM AcH. Effects were additive at that AcH concentration with heparin6k. At the lower (22.3 mM) AcH concentration, notable synergism was seen with heparin3k and with CSC, except with the [(CSC þ AcH) þ FIXa] mixture (which was additive). The remaining effects were essentially additive. Additive effects are interpreted as suggesting that AcH affected the enzyme by adduct formation, altering its 3-dimensional structure and stimulating reactivity of the enzyme with Antithrombin III (ATIII). Similarly, the GAG might bind to the enzyme at its allosteric site thereby increasing its reactivity with ATIII in a manner analogous to that of the 15-fold

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Fig. 7. Effect of acetaldehyde (AcH) on Factor IXa (FIXa) and 25 mg Chondroitin Sulfate A (CSA) (Error bars represent standard error of the mean). Fig. 6. Effect of acetaldehyde (AcH) on Factor IXa (FIXa) and 1 mg Chondroitin Sulfate B [CSB, Dermatan Sulfate (DS)] (Error bars represent standard error of the mean).

stimulation of the reaction of thrombin with ATIII by heparin, as reported by Rosenberg (1985). The synergism observed upon interaction of AcH and GAGs with FIXa, an enzyme associated with the intrinsic blood clotting system, parallels the synergism reported in reactions of 44.3 and 443 mM AcH with various heparins and FXa (Brecher and Hommema, 2002), as well as synergisms noted with Factor II (FII) at the 447mM AcH level, (Brecher and Adamu, 2005), and synergism with a [(heparin17k þ AcH) þ FIIa] pre-mixture at 447 mM AcH (Brecher and Fu, 1999). Lastly, there is confirmation of the synergism in the PT assays when AcH and heparin17k are pre-mixed (Brecher et al., 1999). The mechanism of this consistent synergism is, as yet, elusive. However, a tentative suggestion may be offered that a ternary complex representing cross-linking between the enzyme and the GAG, stabilized by AcH, may promote a more rapid interaction with ATIII. Such a hypothesis remains to be tested in future investigations. Whereas H17k and H6k exhibited a greater effect upon APTT when pre-incubated with FIXa relative to FIXDP, H3k affected a longer APTT upon pre-incubation with

FIXDP. This suggests that H3k also ‘‘hits’’ coagulation factors or their zymogens down the pathway (into the common pathway) in a significant way. Additionally, greater clotting times were seen when 100 mg CSA or 50 mg CSC were preincubated with FIXDP than FIXa. These results suggest that the (high) levels of each of the individual GAGs result in an inactivation of additional coagulation factors found in the FIXDP such that the overall effect is more profound. Acetaldehyde is a highly reactive molecule. Many laboratories have reported the formation of adducts between AcH and proteins, of which only a few are cited here (Clot et al., 1995; Nair et al., 1994; Tuma & Klassen, 1992; Vitala et al., 1997; Wickramasinghe et al., 1994a, 1994b; Worrall et al., 1994, 1996). However the earliest published record of the reaction of aldehydes, that is, formaldehyde, with nucleophiles, including polypeptides is the study by Henriques and Sorensen (1909). The AcH adducts within the organism have led to the development of antibodies there too. With the instantaneous reactivity of AcH with nucleophiles, only a minute amount of AcH will be available as free AcH. Hence, the preponderance of AcH is observed as adducts to proteins (reviewed in Brecher, 2005). Alcoholics generally exhibit very low micromolar levels of AcH freely circulating in their systems. However, it might be as high as 30 mM (Hatake et al., 1990). Nonetheless, free

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that of the daily presence of AcH in severe alcoholics. Clearance rates of modified proteins vary with each protein. Hence, an alcoholic might conceivably have a considerable quantity of AcH adducts retained in his/her system. The GAG-AcH effects upon coagulation observed in this study suggest the necessity of exploring dynamics involved in a number of pathological conditions where altered levels of GAGs have been reported. These include Costello Syndrome and Hurler Disease (Hinek et al., 2004, 2005), Parkinson’s Disease (Kurup and Kurup, 2003), osteoarthritis (Uesake et al., 2001), prostate cancer (Ricciardelli et al., 1999), coronary artery disease and thrombotic stroke (Kumari et al., 1995), and diabetes (Ramamurthi et al., 1990), to name a sampling. The GAG effects are of further importance since they have such varied functions as sharing in regulation of cell division, migration, and proliferation (Karousou et al., 2005), lipoprotein uptake (Rapp and Huttinger, 2005), and in clinical treatment for osteoarthritis Stages 1e4 by Kellgren-Laurence (Lazebnik and Drozdov, 2005). Accordingly, the influence of AcH-GAG dynamics on an increasing number of conditions remains to be explored.

Fig. 8. Effect of acetaldehyde (AcH) on Factor IXa (FIXa) and 10 mg Chondroitin Sulfate C (CSC) (Error bars represent standard error of the mean).

AcH in two Japanese alcoholics were reported as 750 and 2,410 mg/dl, equating to 171 and 548 mM (Watanabe et al., 1985). Whereas AcH reacts instantaneously with nucleophiles, some of its interactions are reversible (Hernandez-Munoz et al., 1992; Israel et al., 1992). Accordingly, it is difficult to predict the level of circulating (free) AcH in alcoholics at any given time. It is known that French males can consume 560 g ethanol daily (Lelbach, 1976), corresponding to 12.2 mol daily, and that humans metabolize 10 ml of ethanol per hour (Goldstein et al., 1968). Salaspuro (1991) suggests that chronic alcoholics might double the rate of oxidation of ethanol. Therefore, the alcoholic is continuously metabolizing ethanol and acetaldehyde. More than 90% of the ethanol is oxidized to CO2 and H2O. Approximately 5% of acetaldehyde produced reacts with available proteins and other nucleophiles. Hence, 0.61 mol of the 12.2 mol of ingested ethanol in a given day may remain as AcH and react with the nucleophiles. Assuming that a 70 kg man contains 41 l H2O, then 0.61 mol/41 l equates to approximately 15 mM/l of AcH in fluids. This number may not be a true value since AcH can react with particulate elements within the cells to form adducts. On the basis of the total 70 kg mass, the AcH concentration might be reduced to 8.7 mM for a 24-h period. The 22.3 mM AcH employed in this study ‘‘approaches’’

Acknowledgments The authors gratefully express their appreciation to the Ohio-West Virginia Affiliate of the American Heart Association and to the NSF (for an REU grant to support KDG) for their support.

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