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N -ACETYL-CYSTEINE REDUCES HOMOCYSTEINE PLASMA LEVELS AFTER SINGLE INTRAVENOUS ADMINISTRATION BY INCREASING THIOLS URINARY EXCRETION
Pharmacological Research, Vol. 40, No. 4, 1999 Article No. phrs.1999.0519, available online at http:rrwww.idealibrary.com on
N-ACETYL-CYSTEINE REDUCES HOMOCYSTEINE PLASMA LEVELS AFTER SINGLE INTRAVENOUS ADMINISTRATION BY INCREASING THIOLS URINARY EXCRETION PAOLO VENTURAU , ROSSANA PANINI, MARIA CRISTINA PASINI, GABRIELLA SCARPETTA and GIANFRANCO SALVIOLI Department of Internal Medicine, Chair of Geriatrics and Gerontology, Uni¨ ersity of Modena and Reggio Emilia, V.le V.Veneto 9, 41100 Modena, Italy Accepted 8 April 1999
A decrease of plasma homocysteine ŽHcy. may represent a therapeutic promise for reducing the impact of atherosclerosis. N-Acetyl-cysteine ŽNAC. is a thiol-containing compound interfering with endogenous thiols, cysteine ŽCys. and Hcy, by forming with them mixed disulphides with a possibly more efficient renal clearance. The aim of this work was to assess the effect of NAC intravenous infusion on plasma levels of different forms of Hcy and particularly to verify the effect on Hcy renal excretion. We collected basal blood samples at 0.5, 1, 2, 5, 8 and 24 h after the beginning of NAC infusion Ž50 mg kgy1 body wt.. and also 24-h urine samples of the day of NAC infusion and of the day before and of the day after the infusion in ten healthy subjects Žmean age 73 " 15.. Urinary and plasma thiols ŽHcy, Cys and NAC. were assayed by HPLC. Both total plasma Hcy Žapprox. 69% ¨ s basal values. and Cys Žapprox. 40% ¨ s basal values. fell progressively, reaching a minimum 5 h after infusion start; total free Ži.e. not bound to proteins. Hcy Ž2.2" 1.8 down from 4.4" 4.2 nmol mly1 . and Cys Ž70.4" 39.8 down from 113.3" 61.2 nmol mly1 . decreased as well. Reduced Žthiolic-free form. Hcy and Cys decreased during infusion, though not as pronounced as for the other forms. Percentagewise, out of the total plasma levels, Hcy and Cys total free form and reduced form tended to increase over infusion as well as their difference Ži.e. the plasma mixed disulphide moiety., thus supporting the idea that excess NAC displaces thiols from their plasma binding sites forming mixed disulphides. Urinary total Cys and Hcy excretion significantly increased at the end of the day of NAC infusion Žtenfold for Cys and fivefold for Hcy. and reduced appreciably on the following day. Also urinary excretion of the free form of Cys and Hcy increased at the end of the day of NAC infusion, although in a lower amount with respect of total amounts, meaning a reduction of percentage Cys and Hcy excreted as the free form; for none of the patients had proteinuria, the ‘free’ form of urine thiols has to be identified in the ‘reduced’ form, the difference between the total and free form reflecting the ‘mixed disulphide’ moiety. NAC intravenous administration induces an efficient and rapid reduction of plasma thiols, particularly of Hcy; our data support the hypothesis that NAC displaces thiols from their binding protein sites and forms, in excess of plasma NAC, mixed disulphides ŽNAC᎐Hcy. with an high renal clearance. This effect may represent the start of an alternative approach in the treatment of hyperhomocysteinaemic conditions. 䊚 1999 Academic Press KEY
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WORDS:
homocysteine, N-acetylcysteine ŽNAC., mixed disulphide.
Corresponding author.
1043᎐6618r99r100345᎐06r$30.00r0
䊚 1999 Academic Press
346
INTRODUCTION
Homocysteine ŽHcy. is a sulphydryl-containing aminoacid derived from the metabolism Ždemethylation. of methionine. In human plasma, approximately 20᎐30% of total Hcy circulates as the free form Žmostly, approx. 20᎐30% out of total Hcy, as ‘oxidised form’, i.e. disulphide form: homocysteine or ‘mixed disulphide’ homocysteine᎐cysteine; only a smaller amount, approx. 1᎐2% out of total Hcy, as the ‘reduced form’, i.e. Hcy in thiolic form., whereas the major portion Ž70᎐80% of total Hcy. circulates as protein-bound form by the formation of mixed disulphide with thiolic lateral residues of circulating proteins, particularly albumin w1᎐3x. Free and protein-bound forms in plasma are collectively designated as homocystŽe.ine wHŽe.x. Greatly elevated plasma levels Žapprox. 200 nmol mly1 . of total HŽe. are found in subjects affected by homocystinuria; these patients exhibit early atherosclerosis disease as well as arterial and venous thrombosis w4᎐6x. More recently even mild hyperhomocysteinaemia Žovernight fasting plasma levels ) 15 nmol mly1 . has been recognized as a risk factor for vascular disease w7᎐9x. Many experimental works demonstrate mild hyperhomocysteinaemia as an independent risk factor for vascular disease w10x, even if the underlying mechanism of vascular damage is not yet well known w11x. Thus, reduction of total plasma homocysteine levels may hold therapeutic promise for reducing the impact of atherosclerotic disease and may represent an important therapeutical approach for hyperhomocysteinaemic patients. N-Acetyl-cysteine ŽNAC. is a thiol-containing compound widely used for many years as a mucolytic drug, particularly in the treatment of chronical obstructive pulmonary disease ŽCOPD. w12, 13x. More recently, NAC has been used in the treatment of paracetamol poisoning w14᎐16x. In both cases the drug has been shown to be relatively well tolerated and without serious side effects w13᎐16x. Its mucolytic effect is supposed to be mediated by the splicing of disulphide bounds between mucus macromolecules w17, 18x. Another effect of NAC may be an interference with the metabolism of thiol compounds in plasma. The presence of a sulphydryl group in NAC explains its tendency to bind to cysteine ŽCys. and other endogenous sulphydryl-containing compounds. Studies in rats have demonstrated that chronic NAC administration increases the total amount of reduced Cys and thus changes the redox status in plasma w18x. It also displaces cysteine or other thiols from their protein binding sites, forming various mixed disulphides, including protein low molecular-weight disulphides w18, 19x. It has been suggested that the low molecular weight mixed disulphides thus formed with NAC have a
Pharmacological Research, Vol. 40, No. 4, 1999
high renal clearance and possibly also increased metabolic bioavailability, which may lead to a more efficient elimination of thiols, and particularly Hcy, from plasma. The aim of this work was to evaluate plasma and urine Hcy and Cys levels after single intravenous NAC administration; particularly, it was our aim to verify the effect of NAC intravenous infusion on total, total free and ‘reduced’ plasma levels of Hcy and assess its effect on Hcy renal excretion. If Hcy NAC-induced reduction is confirmed and these results will be supported by further trials on chronic treatment, this may represent the starting point for a new therapeutical approach, as associated or alternative treatment, for hyperhomocysteinaemic conditions.
MATERIALS AND METHODS Ten subjects Žtwo females, mean age 73 " 15. were consecutively studied. High Ž) 15 nmol mly1 . basal Hcy levels were not considered an exclusion criteria. None of them had significantly altered liver or renal function, nor were they affected by COPD or had detectable proteinuria. Informed consent was obtained from all subjects. After a 2-day wash-out period Žstandard diet., on the morning of the third day, after overnight fasting, a 5-ml blood sample was collected for determination of basal plasma thiols concentration; at the same time we started a 24-h urine collection for determination of basal daily urinary Cys, Hcy and NAC excretion. On the following morning, after an overnight fast, we collected a blood sample for another determination of plasma thiol concentrations, and then the subject underwent an intravenous NAC infusion Ž50 mg kgy1 body wt. in saline 500 ml in approx. 1 h.. Following intravenous NAC, we took 5-ml blood samples at 0.5, 1, 2, 5, 8 and 24 h after the beginning of infusion and started the 24-h urine collection, which was kept both for the day of intravenous NAC administration and for the following day. In each urine sample we also assayed creatinine content w20x, in order to evaluate urinary excretion of thiols as homocysteine ᎏor cysteinercreatinine ratio Žnmol mgy1 creatinine. and protein content w21x. The different urine and plasma thiol forms ŽHcy, Cys and NAC. were assayed by a HPLC method with fluorimetric detection according to Cornwell et al. w22x, using cysteamine as internal standard, to improve quantitative determination. Each blood sample was collected in a polypropylene tube containing 100 l of 10% EDTA solution and placed immediately on ice and centrifuged at 4000 g at 4⬚C for 5 min. Approximately 900 l of plasma Ž300 l each. were used to assay each different form Žreduced,
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total free and total. of plasma Hcy and Cys. Total plasma forms were obtained according to the preparation steps described by Cornwell w22x; reduced forms were obtained by immediate derivatization of plasma with 7-fluoro-benzo-2-oxa-1,3-diazole-4sulphonic acid ŽSBD-F., followed by precipitation of plasma protein with trichloroacetic acid ŽTCA.. Total free forms were obtained by immediate plasma protein precipitation with TCA, followed by a reduction step with tri-n-butylphosphine and derivatization step with SBD-F. Ten millilitres of 24-h urine collection Žpreserved with 15% sulphosalicilic acid solution. were immediately treated as plasma or stored at y70⬚C until analysis. The results are expressed as means " SD. Statistical significance Ž P- 0.05. between groups was tested by two-way ANOVA for dependent samples using a SPSS 䊛 commercial package.
RESULTS Table I summarises biochemical and anthropometrical data of studied subjects. Cys and Hcy plasma levels of 2 consecutive days in each single patient were not significantly different. No patients showed adverse reactions or intolerance symptoms during NAC infusion. Tables II᎐IV show the mean " SD values of different forms Žtotal, free and reduced. of NAC, Cys and Hcy, respectively, during NAC infusion. As shown in Table IV Žmean values " SD of ten patients., NAC plasma concentrations rose during intravenous NAC infusion, reaching maximal values at the second hour of infusion Žmean values 561 " 250 nmol mly1 ., then progressively decreasing until nearly disappearing 24 h after the beginning of infusion Ž11.4" 8.1 nmol mly1 .; after infusion, the levels of total plasma Cys fell progressively, reaching minimum values between the second and fifth hour after the starting of infusion Ž150 " 57 nmol mly1 ¨ s 262.7 nmol mly1 ., a reduction of approximately 40% from baseline values. During NAC infusion, even
Table I Biochemicals data of studied subjectsU Parameter
Range
Age Žyears. Albumin Žg dly1 . Total plasma protein Žg dly1 . Haemoglobin Žg dly1 . Lymphocyte count Žn mly1 . PCR Žmg dly1 . VES Žmm hy1 . Total cholesterol Žmg dly1 . Tryglycerides Žmg dly1 . Creatinine clearance Žml miny1 . Urinary protein Žmg diey1 . U
73.4" 14.5 3.84" 0.3 6.88" 0.4 14.54" 1.5 2336.9" 734.2 0.51" 0.5 12.6" 3.1 217.7" 24.9 191 " 120 69.2" 18.7 4.5" 3.1
Values are expressed as mean " SD.
total plasma Hcy levels fell progressively, from the baseline values of 22.46" 17.6 nmol mly1 to a minimum of 6.69" 4.96 nmol mly1 5 h after the beginning of infusion, with a reduction of approximately 69% with respect of basal mean value. Plasma Cys and Hcy levels then rose progressively, though not reaching the initial values, 24 h after the end of infusion Ž239 " 97 for Cys and 16.46" 12.9 nmol mly1 for Hcy.. Considering the total free and the reduced forms of these substances ŽTables II and III., during NAC infusion the absolute value of the free Ži.e. not bound to proteins. Hcy and Cys tended to fall, reaching the lowest values 5᎐8 h after the start of infusion Ž70.4" 39.8 nmol mly1 , down from 113.3" 61.2 nmol mly1 for Cys, and 2.2" 1.8 nmol mly1 down from 4.4" 4.2 nmol mly1 for Hcy.. The absolute values of the reduced forms of Hcy and Cys also decreased during infusion ŽTables II and III., though not as pronounced as for the other values, reaching minimum values approximately 5 h after infusion. Percentagewise, out of the total plasma levels, Hcy and Cys total free and reduced forms tended to increase over infusion, as well the difference between the free and reduced amount Ži.e. the mixed disulphide plasma moiety. reaching the highest values 1᎐2 h after infusion, in concomitance with the peak of plasma NAC levels.
Table II Different fractions of plasma acetylcysteine during and after 60 min NAC endovenous admnistrationU Basal
30 min
60 min
120 min
300 min
480 min
24 h
T-NAC
n.d.
182.1" 63.3
377.7" 91.6
561.7" 250.4
188.1" 95.2
99.8" 53.5
11.4" 12.44
TF-NAC
n.d
94.2" 54.4 Ž52%.
184.6" 64.6 Ž48%.
312.4" 149 Ž56%.
99.8" 42.9 Ž53%.
39.3" 22.6 Ž40%.
5.2" 2.3 Ž46%.
R-NAC
n.d.
9.1" 5.3 Ž10%.
56.5" 12.4 Ž14%.
95.37" 20.1 Ž17%.
16.7" 2.4 Ž7%.
7.2" 2.4 Ž7%.
n.d.
U
Values are expressed as mean values Žnmol mly1 . " SD; values in brackets express percent of total. T-NAC, total N-acetylcysteine; TF-NAC, total free N-acetylcysteine; R-NAC, reduced N-acetylcysteine.
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348
Table III Different fractions of plasma cysteine during and after 60 min NAC endovenous administrationU Basal
30 min
60 min
120 min
300 min
480 min
24 h
T-Cys
262.7" 104.5
199.6" 65.1
176.2" 65.1
151.7" 57.1
149 " 61.1
157.4" 49.8
239.9" 97.5
TF-Cys
113.3" 61.2 Ž43%.
96.9" 33.2 Ž48%.
102.5" 42.3 Ž58%.
95.1" 47.1 Ž63%.
75.4" 45.2 Ž50%.
70.4" 39.8 Ž45%.
90.8" 51.4 Ž38%.
R-Cys
7.82" 1.1 Ž2.9%.
5.99" 0.8 Ž3.0%.
5.79" 1.0 Ž3.2%.
5.99" 1.1 Ž3.9%.
5.04" 0.7 Ž3.3%.
4.86" 0.8 Ž3.0%.
7.2" 0.7 Ž3.0%.
U Values are expressed as mean Žnmol mly1 . values " SD; values in brackets express percent of total. T-Cys, total cysteine; TF-Cys, total free cysteine; R-Cys, reduced cysteine.
The urinary parameters are expressed in terms of thiol ŽCys or Hcy. concentration Žnmol. per milligram of urinary creatinine ŽTable V.. As compared to pre-infusion values, urinary excretion of total Cys and Hcy increased in a significant manner by the end of the first day of NAC infusion Žapprox. tenfold for Cys and fivefold for Hcy.; instead, on the day following the infusion, urinary excretion of total amount of these thiols reduced appreciably, almost reaching pre-infusion values. NAC behaved similarly; it was found in very scanty amounts in basal samples Žcysteine metabolism. w12, 23x, in very high amounts Žapprox. 100-fold. in the 24-h urine of infusion day and it decreased remarkably on the day after infusion. Also the urinary amount of total free forms of Cys and Hcy showed an increase on the day of NAC infusion, although it was lower with respect of corresponding total forms, thus resulting in a reduction of percentage of Cys and Hcy excreted as free form.
DISCUSSION Our data demonstrate that intravenous administration of NAC Ž50 mg kgy1 body wt.. induces a rapid and important reduction of both Hcy Žby approx. 55᎐69% of the basal total levels 2 and 5 h after the start of infusion., and cysteine plasma levels Žby approx. 35᎐45% fall over the same period.. However, this effect tended to diminish over time: 24 h
after the end of infusion the plasma thiol levels tended to return to baseline, remaining slightly below the basal values. The reduction of the absolute plasma levels of both total and free fractions Žtotal free and reduced thiols. and the percentage increase both of the total free Žand thus reduction of the protein-bound fraction. and of the reduced form out of the total amount ŽTables III and IV., support the hypothesis that NAC infusion alters the protein-binding of thiol components in plasma, probably by displacing circulating thiols from their protein binding sites w18x. Moreover, the percentage increase of total free forms and the concurrent lower increase of the reduced forms suggest an increase of mixed disulphide Ži.e. difference between total free and reduced moiety. formation Žlikely as acetylcysteine ᎐homocysteine or acetylcysteine ᎐cysteine mixed disulphide.. Considering urinary parameters, our data demonstrate a significant increase of renal thiols excretion induced by NAC administration. According to the literature w12, 23x, we observed a little amount of daily NAC excretion Žendogenous NAC. ŽTable V, first column. as expression of normal cysteine metabolism and an increase of NAC renal excretion Žapprox. tenfold the normal endogenous amount. on the day of infusion, showing a relative quick renal clearance. According to our data, both Cys and Hcy renal excretion increase by a very significant amount on the day of NAC infusion, in correspondence of higher NAC excretion ŽTable V..
Table IV Different fractions of plasma homocysteine during and after 60 min NAC endovenous administrationU Basal
30 min
60 min
120 min
300 min
480 min
24 h
22.4" 17.7
16.4" 13.1
13.1" 11.8
10.0" 8.9
6.9" 4.6
9.3" 5.7
16.4" 12.9
TF-Hcy
4.4" 4.3 Ž20%.
5.9" 5.4 Ž36%.
6.2" 6.0 Ž47%.
4.5" 4.1 Ž45%.
2.6" 2.6 Ž37%.
2.2" 1.8 Ž24%.
4.1" 3.5 Ž25%.
R-Hcy
0.36" 0.11 Ž1.6%.
0.35" 0.12 Ž2.1%.
0.26" 0.20 Ž1.9%.
0.28" 0.11 Ž2.8%.
0.19" 0.09 Ž2.7%.
0.20" 0.1 Ž2.1%.
0.24" 0.18 Ž1.5%.
T-Hcy
U
Values are expressed as mean values Žnmol mly1 . " SD; values in brackets express percent of total. T-Hcy, total homocysteine; TF-Hcy, total free homocysteine; R-Hcy, reduced homocysteine.
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349
Table V Urinary thiols excretion† Day I T-Cys
Day II
Day III
165.67" 51.6 1076.65" 363.7U U
160.48" 62.3‡
TF-Cys
83.83" 40.1 Ž50%.
390.66" 281.5 Ž36%.
98.39" 61.5‡ Ž61.2%.
T-Hcy
13.26" 9.0
50.17" 35.2U
19.27" 19.0‡
U
4.82" 2.61 ‡ Ž25%.
TF-Hcy
3.83" 1.1 Ž29%.
10.27" 7.35 Ž20%.
T-NAC
3.15" 1.5
2817.33" 1600.4U
TF-NAC
n.d.
U
1245.63" 1047.9 Ž44%.
28.73" 21.7 11.61" 9.7 Ž40%.
†Values are expressed Žmean " SD. as nmol mgy1 of urinary creatinine. Values in parentheses express percent of total. T-Cys, total cysteine; TF-Cys, total free cysteine; T-Hcy, total homocysteine; TF-Hcy, total free homocysteine; T-NAC, total N-acetylcysteine; TF-NAC, total free N-acetylcysteine; ‡not statistically significant with respect of corresponding basal values; U statistically Ž P- 0.01. significant from corresponding basal values. Day I, urinary levels of the day before infusion; Day II, urinary levels of the day of infusion; day III, urinary levels of the day after infusion.
Also, urinary free forms of Cys and Hcy increase on the day of NAC infusion, though to a lower extent with respect to the total corresponding amount, this indicates a reduction of percentage of Cys and Hcy excreted as the free form. Since none of the patients had significant proteinuria ŽTable I., the ‘free’ form of urine thiols can be identified in the ‘reduced’ form: thus in urine the difference between the total and free form reflects the ‘mixed disulphide’ amount. These data are consistent with a higher renal NAC-induced excretion of thiols as mixed disulphides Žprobably NAC᎐cysteine and NAC᎐homocysteine mixed disulphide.. Nevertheless, it has to be noticed that the absolute increase of Hcy and Cys excreted as the reduced form on the day of NAC infusion may also reflect a competition of NAC excess for tubular reabsorption sites w24x. In conclusion, a single NAC intravenous administration induces an efficient and rapid decrease of the main circulating plasma thiols ŽCys and Hcy.. This effect seems particularly relevant for Hcy and may be partly explained by displacement by NAC of thiols from their plasma binding sites Žplasma proteins. and by the formation, in large excess of plasma NAC, of NAC᎐Cys and NAC᎐Hcy mixed disulphides, which in turn undergoes a higher renal clearance, resulting in an increase of urinary excretion, likely as mixed disulphide forms. This approach may be of interest in decreasing plasma levels of atherogenic aminothiol homocysteine, particularly as an association therapy or as an
alternative approach in hyperhomocysteinaemic conditions poorly sensitive to vitamin administration w25, 26x. NAC may also have other important protective effects against Hcy toxicity. Its antioxidant capacity is well known, particularly against hydrogen peroxide w27x, an important intermediate in Hcy-induced toxicity w28x. Moreover, according to our data, NAC administration seems to decrease the plasma levels of the reduced form of Hcy, the one available to transformation in homocysteine᎐thiolactone, one of the most toxic Hcy metabolites w28x; from this point of view also, an increase of mixed-disulphide formation may represent a positive effect. As already mentioned, the effect of intravenous single infusion seems to be transient Žthe thiol levels tend to return to basal values on the day following the infusion.; this fact, together with the observed reduction of cysteine levels, is not a favourable effect: thus chronical administration studies, particularly about NAC oral administration, are needed before making conclusions about its effectiveness in the treatment of hyperhomocysteinaemic conditions.
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Masson P, Hultberg B. N-Acetylcysteine treatment lowers plasma homocysteine but not serum lipoproteinŽa. levels. Atherosclerosis 1996; 119: 99᎐106. Bartels H, Bohmer M, Heierli C. Serum creatinine determination without protein precipitation. Clin Chem Acta 1972; 37: 193᎐7. Lowry OH, Rosebrough NJ, Farr P, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265᎐75. Cornwell PE, Morgan SL, Vaughn WH. Modification of a high performance liquid chromatographic method for assay of homocysteine in human plasma. J Chromatogr 1993; 617: 136᎐9. Rodenstein D, De Coster A, Gazzaniga A. Pharmacokinetics of oral acetylcysteine: absorption, binding and metabolism in patients with respiratory disorders. Clin Pharmacokinet 1978; 3: 247᎐54. Foreman JW, Wald H, Blumberg G, Pepe LM, Segal S. Homocysteine uptake in isolated rat renal cortical tubules. Metabolism 1982; 31: 617᎐19. Kang SS. Treatment of hyperhomocystŽe.inemia: physiological basis. J Nutr 1996; 126: 1273S᎐5S. Brattstrom ¨ L. Vitamins as homocysteine-lowering agents. J Nutr 1996; 126: 1276S᎐80S Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radical Bio Med 1989; 6: 593᎐7. Olszewski AJ, McCully K. Homocysteine and the oxidative modification of proteins and lipids. Free Radical Bio Med 1993; 14: 683᎐93.
N-ACETYL-CYSTEINE REDUCES HOMOCYSTEINE PLASMA LEVELS AFTER SINGLE INTRAVENOUS ADMINISTRATION BY INCREASING THIOLS URINARY EXCRETION PAOLO VENTURAU , ROSSANA PANINI, MARIA CRISTINA PASINI, GABRIELLA SCARPETTA and GIANFRANCO SALVIOLI Department of Internal Medicine, Chair of Geriatrics and Gerontology, Uni¨ ersity of Modena and Reggio Emilia, V.le V.Veneto 9, 41100 Modena, Italy Accepted 8 April 1999
A decrease of plasma homocysteine ŽHcy. may represent a therapeutic promise for reducing the impact of atherosclerosis. N-Acetyl-cysteine ŽNAC. is a thiol-containing compound interfering with endogenous thiols, cysteine ŽCys. and Hcy, by forming with them mixed disulphides with a possibly more efficient renal clearance. The aim of this work was to assess the effect of NAC intravenous infusion on plasma levels of different forms of Hcy and particularly to verify the effect on Hcy renal excretion. We collected basal blood samples at 0.5, 1, 2, 5, 8 and 24 h after the beginning of NAC infusion Ž50 mg kgy1 body wt.. and also 24-h urine samples of the day of NAC infusion and of the day before and of the day after the infusion in ten healthy subjects Žmean age 73 " 15.. Urinary and plasma thiols ŽHcy, Cys and NAC. were assayed by HPLC. Both total plasma Hcy Žapprox. 69% ¨ s basal values. and Cys Žapprox. 40% ¨ s basal values. fell progressively, reaching a minimum 5 h after infusion start; total free Ži.e. not bound to proteins. Hcy Ž2.2" 1.8 down from 4.4" 4.2 nmol mly1 . and Cys Ž70.4" 39.8 down from 113.3" 61.2 nmol mly1 . decreased as well. Reduced Žthiolic-free form. Hcy and Cys decreased during infusion, though not as pronounced as for the other forms. Percentagewise, out of the total plasma levels, Hcy and Cys total free form and reduced form tended to increase over infusion as well as their difference Ži.e. the plasma mixed disulphide moiety., thus supporting the idea that excess NAC displaces thiols from their plasma binding sites forming mixed disulphides. Urinary total Cys and Hcy excretion significantly increased at the end of the day of NAC infusion Žtenfold for Cys and fivefold for Hcy. and reduced appreciably on the following day. Also urinary excretion of the free form of Cys and Hcy increased at the end of the day of NAC infusion, although in a lower amount with respect of total amounts, meaning a reduction of percentage Cys and Hcy excreted as the free form; for none of the patients had proteinuria, the ‘free’ form of urine thiols has to be identified in the ‘reduced’ form, the difference between the total and free form reflecting the ‘mixed disulphide’ moiety. NAC intravenous administration induces an efficient and rapid reduction of plasma thiols, particularly of Hcy; our data support the hypothesis that NAC displaces thiols from their binding protein sites and forms, in excess of plasma NAC, mixed disulphides ŽNAC᎐Hcy. with an high renal clearance. This effect may represent the start of an alternative approach in the treatment of hyperhomocysteinaemic conditions. 䊚 1999 Academic Press KEY
U
WORDS:
homocysteine, N-acetylcysteine ŽNAC., mixed disulphide.
Corresponding author.
1043᎐6618r99r100345᎐06r$30.00r0
䊚 1999 Academic Press
346
INTRODUCTION
Homocysteine ŽHcy. is a sulphydryl-containing aminoacid derived from the metabolism Ždemethylation. of methionine. In human plasma, approximately 20᎐30% of total Hcy circulates as the free form Žmostly, approx. 20᎐30% out of total Hcy, as ‘oxidised form’, i.e. disulphide form: homocysteine or ‘mixed disulphide’ homocysteine᎐cysteine; only a smaller amount, approx. 1᎐2% out of total Hcy, as the ‘reduced form’, i.e. Hcy in thiolic form., whereas the major portion Ž70᎐80% of total Hcy. circulates as protein-bound form by the formation of mixed disulphide with thiolic lateral residues of circulating proteins, particularly albumin w1᎐3x. Free and protein-bound forms in plasma are collectively designated as homocystŽe.ine wHŽe.x. Greatly elevated plasma levels Žapprox. 200 nmol mly1 . of total HŽe. are found in subjects affected by homocystinuria; these patients exhibit early atherosclerosis disease as well as arterial and venous thrombosis w4᎐6x. More recently even mild hyperhomocysteinaemia Žovernight fasting plasma levels ) 15 nmol mly1 . has been recognized as a risk factor for vascular disease w7᎐9x. Many experimental works demonstrate mild hyperhomocysteinaemia as an independent risk factor for vascular disease w10x, even if the underlying mechanism of vascular damage is not yet well known w11x. Thus, reduction of total plasma homocysteine levels may hold therapeutic promise for reducing the impact of atherosclerotic disease and may represent an important therapeutical approach for hyperhomocysteinaemic patients. N-Acetyl-cysteine ŽNAC. is a thiol-containing compound widely used for many years as a mucolytic drug, particularly in the treatment of chronical obstructive pulmonary disease ŽCOPD. w12, 13x. More recently, NAC has been used in the treatment of paracetamol poisoning w14᎐16x. In both cases the drug has been shown to be relatively well tolerated and without serious side effects w13᎐16x. Its mucolytic effect is supposed to be mediated by the splicing of disulphide bounds between mucus macromolecules w17, 18x. Another effect of NAC may be an interference with the metabolism of thiol compounds in plasma. The presence of a sulphydryl group in NAC explains its tendency to bind to cysteine ŽCys. and other endogenous sulphydryl-containing compounds. Studies in rats have demonstrated that chronic NAC administration increases the total amount of reduced Cys and thus changes the redox status in plasma w18x. It also displaces cysteine or other thiols from their protein binding sites, forming various mixed disulphides, including protein low molecular-weight disulphides w18, 19x. It has been suggested that the low molecular weight mixed disulphides thus formed with NAC have a
Pharmacological Research, Vol. 40, No. 4, 1999
high renal clearance and possibly also increased metabolic bioavailability, which may lead to a more efficient elimination of thiols, and particularly Hcy, from plasma. The aim of this work was to evaluate plasma and urine Hcy and Cys levels after single intravenous NAC administration; particularly, it was our aim to verify the effect of NAC intravenous infusion on total, total free and ‘reduced’ plasma levels of Hcy and assess its effect on Hcy renal excretion. If Hcy NAC-induced reduction is confirmed and these results will be supported by further trials on chronic treatment, this may represent the starting point for a new therapeutical approach, as associated or alternative treatment, for hyperhomocysteinaemic conditions.
MATERIALS AND METHODS Ten subjects Žtwo females, mean age 73 " 15. were consecutively studied. High Ž) 15 nmol mly1 . basal Hcy levels were not considered an exclusion criteria. None of them had significantly altered liver or renal function, nor were they affected by COPD or had detectable proteinuria. Informed consent was obtained from all subjects. After a 2-day wash-out period Žstandard diet., on the morning of the third day, after overnight fasting, a 5-ml blood sample was collected for determination of basal plasma thiols concentration; at the same time we started a 24-h urine collection for determination of basal daily urinary Cys, Hcy and NAC excretion. On the following morning, after an overnight fast, we collected a blood sample for another determination of plasma thiol concentrations, and then the subject underwent an intravenous NAC infusion Ž50 mg kgy1 body wt. in saline 500 ml in approx. 1 h.. Following intravenous NAC, we took 5-ml blood samples at 0.5, 1, 2, 5, 8 and 24 h after the beginning of infusion and started the 24-h urine collection, which was kept both for the day of intravenous NAC administration and for the following day. In each urine sample we also assayed creatinine content w20x, in order to evaluate urinary excretion of thiols as homocysteine ᎏor cysteinercreatinine ratio Žnmol mgy1 creatinine. and protein content w21x. The different urine and plasma thiol forms ŽHcy, Cys and NAC. were assayed by a HPLC method with fluorimetric detection according to Cornwell et al. w22x, using cysteamine as internal standard, to improve quantitative determination. Each blood sample was collected in a polypropylene tube containing 100 l of 10% EDTA solution and placed immediately on ice and centrifuged at 4000 g at 4⬚C for 5 min. Approximately 900 l of plasma Ž300 l each. were used to assay each different form Žreduced,
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347
total free and total. of plasma Hcy and Cys. Total plasma forms were obtained according to the preparation steps described by Cornwell w22x; reduced forms were obtained by immediate derivatization of plasma with 7-fluoro-benzo-2-oxa-1,3-diazole-4sulphonic acid ŽSBD-F., followed by precipitation of plasma protein with trichloroacetic acid ŽTCA.. Total free forms were obtained by immediate plasma protein precipitation with TCA, followed by a reduction step with tri-n-butylphosphine and derivatization step with SBD-F. Ten millilitres of 24-h urine collection Žpreserved with 15% sulphosalicilic acid solution. were immediately treated as plasma or stored at y70⬚C until analysis. The results are expressed as means " SD. Statistical significance Ž P- 0.05. between groups was tested by two-way ANOVA for dependent samples using a SPSS 䊛 commercial package.
RESULTS Table I summarises biochemical and anthropometrical data of studied subjects. Cys and Hcy plasma levels of 2 consecutive days in each single patient were not significantly different. No patients showed adverse reactions or intolerance symptoms during NAC infusion. Tables II᎐IV show the mean " SD values of different forms Žtotal, free and reduced. of NAC, Cys and Hcy, respectively, during NAC infusion. As shown in Table IV Žmean values " SD of ten patients., NAC plasma concentrations rose during intravenous NAC infusion, reaching maximal values at the second hour of infusion Žmean values 561 " 250 nmol mly1 ., then progressively decreasing until nearly disappearing 24 h after the beginning of infusion Ž11.4" 8.1 nmol mly1 .; after infusion, the levels of total plasma Cys fell progressively, reaching minimum values between the second and fifth hour after the starting of infusion Ž150 " 57 nmol mly1 ¨ s 262.7 nmol mly1 ., a reduction of approximately 40% from baseline values. During NAC infusion, even
Table I Biochemicals data of studied subjectsU Parameter
Range
Age Žyears. Albumin Žg dly1 . Total plasma protein Žg dly1 . Haemoglobin Žg dly1 . Lymphocyte count Žn mly1 . PCR Žmg dly1 . VES Žmm hy1 . Total cholesterol Žmg dly1 . Tryglycerides Žmg dly1 . Creatinine clearance Žml miny1 . Urinary protein Žmg diey1 . U
73.4" 14.5 3.84" 0.3 6.88" 0.4 14.54" 1.5 2336.9" 734.2 0.51" 0.5 12.6" 3.1 217.7" 24.9 191 " 120 69.2" 18.7 4.5" 3.1
Values are expressed as mean " SD.
total plasma Hcy levels fell progressively, from the baseline values of 22.46" 17.6 nmol mly1 to a minimum of 6.69" 4.96 nmol mly1 5 h after the beginning of infusion, with a reduction of approximately 69% with respect of basal mean value. Plasma Cys and Hcy levels then rose progressively, though not reaching the initial values, 24 h after the end of infusion Ž239 " 97 for Cys and 16.46" 12.9 nmol mly1 for Hcy.. Considering the total free and the reduced forms of these substances ŽTables II and III., during NAC infusion the absolute value of the free Ži.e. not bound to proteins. Hcy and Cys tended to fall, reaching the lowest values 5᎐8 h after the start of infusion Ž70.4" 39.8 nmol mly1 , down from 113.3" 61.2 nmol mly1 for Cys, and 2.2" 1.8 nmol mly1 down from 4.4" 4.2 nmol mly1 for Hcy.. The absolute values of the reduced forms of Hcy and Cys also decreased during infusion ŽTables II and III., though not as pronounced as for the other values, reaching minimum values approximately 5 h after infusion. Percentagewise, out of the total plasma levels, Hcy and Cys total free and reduced forms tended to increase over infusion, as well the difference between the free and reduced amount Ži.e. the mixed disulphide plasma moiety. reaching the highest values 1᎐2 h after infusion, in concomitance with the peak of plasma NAC levels.
Table II Different fractions of plasma acetylcysteine during and after 60 min NAC endovenous admnistrationU Basal
30 min
60 min
120 min
300 min
480 min
24 h
T-NAC
n.d.
182.1" 63.3
377.7" 91.6
561.7" 250.4
188.1" 95.2
99.8" 53.5
11.4" 12.44
TF-NAC
n.d
94.2" 54.4 Ž52%.
184.6" 64.6 Ž48%.
312.4" 149 Ž56%.
99.8" 42.9 Ž53%.
39.3" 22.6 Ž40%.
5.2" 2.3 Ž46%.
R-NAC
n.d.
9.1" 5.3 Ž10%.
56.5" 12.4 Ž14%.
95.37" 20.1 Ž17%.
16.7" 2.4 Ž7%.
7.2" 2.4 Ž7%.
n.d.
U
Values are expressed as mean values Žnmol mly1 . " SD; values in brackets express percent of total. T-NAC, total N-acetylcysteine; TF-NAC, total free N-acetylcysteine; R-NAC, reduced N-acetylcysteine.
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348
Table III Different fractions of plasma cysteine during and after 60 min NAC endovenous administrationU Basal
30 min
60 min
120 min
300 min
480 min
24 h
T-Cys
262.7" 104.5
199.6" 65.1
176.2" 65.1
151.7" 57.1
149 " 61.1
157.4" 49.8
239.9" 97.5
TF-Cys
113.3" 61.2 Ž43%.
96.9" 33.2 Ž48%.
102.5" 42.3 Ž58%.
95.1" 47.1 Ž63%.
75.4" 45.2 Ž50%.
70.4" 39.8 Ž45%.
90.8" 51.4 Ž38%.
R-Cys
7.82" 1.1 Ž2.9%.
5.99" 0.8 Ž3.0%.
5.79" 1.0 Ž3.2%.
5.99" 1.1 Ž3.9%.
5.04" 0.7 Ž3.3%.
4.86" 0.8 Ž3.0%.
7.2" 0.7 Ž3.0%.
U Values are expressed as mean Žnmol mly1 . values " SD; values in brackets express percent of total. T-Cys, total cysteine; TF-Cys, total free cysteine; R-Cys, reduced cysteine.
The urinary parameters are expressed in terms of thiol ŽCys or Hcy. concentration Žnmol. per milligram of urinary creatinine ŽTable V.. As compared to pre-infusion values, urinary excretion of total Cys and Hcy increased in a significant manner by the end of the first day of NAC infusion Žapprox. tenfold for Cys and fivefold for Hcy.; instead, on the day following the infusion, urinary excretion of total amount of these thiols reduced appreciably, almost reaching pre-infusion values. NAC behaved similarly; it was found in very scanty amounts in basal samples Žcysteine metabolism. w12, 23x, in very high amounts Žapprox. 100-fold. in the 24-h urine of infusion day and it decreased remarkably on the day after infusion. Also the urinary amount of total free forms of Cys and Hcy showed an increase on the day of NAC infusion, although it was lower with respect of corresponding total forms, thus resulting in a reduction of percentage of Cys and Hcy excreted as free form.
DISCUSSION Our data demonstrate that intravenous administration of NAC Ž50 mg kgy1 body wt.. induces a rapid and important reduction of both Hcy Žby approx. 55᎐69% of the basal total levels 2 and 5 h after the start of infusion., and cysteine plasma levels Žby approx. 35᎐45% fall over the same period.. However, this effect tended to diminish over time: 24 h
after the end of infusion the plasma thiol levels tended to return to baseline, remaining slightly below the basal values. The reduction of the absolute plasma levels of both total and free fractions Žtotal free and reduced thiols. and the percentage increase both of the total free Žand thus reduction of the protein-bound fraction. and of the reduced form out of the total amount ŽTables III and IV., support the hypothesis that NAC infusion alters the protein-binding of thiol components in plasma, probably by displacing circulating thiols from their protein binding sites w18x. Moreover, the percentage increase of total free forms and the concurrent lower increase of the reduced forms suggest an increase of mixed disulphide Ži.e. difference between total free and reduced moiety. formation Žlikely as acetylcysteine ᎐homocysteine or acetylcysteine ᎐cysteine mixed disulphide.. Considering urinary parameters, our data demonstrate a significant increase of renal thiols excretion induced by NAC administration. According to the literature w12, 23x, we observed a little amount of daily NAC excretion Žendogenous NAC. ŽTable V, first column. as expression of normal cysteine metabolism and an increase of NAC renal excretion Žapprox. tenfold the normal endogenous amount. on the day of infusion, showing a relative quick renal clearance. According to our data, both Cys and Hcy renal excretion increase by a very significant amount on the day of NAC infusion, in correspondence of higher NAC excretion ŽTable V..
Table IV Different fractions of plasma homocysteine during and after 60 min NAC endovenous administrationU Basal
30 min
60 min
120 min
300 min
480 min
24 h
22.4" 17.7
16.4" 13.1
13.1" 11.8
10.0" 8.9
6.9" 4.6
9.3" 5.7
16.4" 12.9
TF-Hcy
4.4" 4.3 Ž20%.
5.9" 5.4 Ž36%.
6.2" 6.0 Ž47%.
4.5" 4.1 Ž45%.
2.6" 2.6 Ž37%.
2.2" 1.8 Ž24%.
4.1" 3.5 Ž25%.
R-Hcy
0.36" 0.11 Ž1.6%.
0.35" 0.12 Ž2.1%.
0.26" 0.20 Ž1.9%.
0.28" 0.11 Ž2.8%.
0.19" 0.09 Ž2.7%.
0.20" 0.1 Ž2.1%.
0.24" 0.18 Ž1.5%.
T-Hcy
U
Values are expressed as mean values Žnmol mly1 . " SD; values in brackets express percent of total. T-Hcy, total homocysteine; TF-Hcy, total free homocysteine; R-Hcy, reduced homocysteine.
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349
Table V Urinary thiols excretion† Day I T-Cys
Day II
Day III
165.67" 51.6 1076.65" 363.7U U
160.48" 62.3‡
TF-Cys
83.83" 40.1 Ž50%.
390.66" 281.5 Ž36%.
98.39" 61.5‡ Ž61.2%.
T-Hcy
13.26" 9.0
50.17" 35.2U
19.27" 19.0‡
U
4.82" 2.61 ‡ Ž25%.
TF-Hcy
3.83" 1.1 Ž29%.
10.27" 7.35 Ž20%.
T-NAC
3.15" 1.5
2817.33" 1600.4U
TF-NAC
n.d.
U
1245.63" 1047.9 Ž44%.
28.73" 21.7 11.61" 9.7 Ž40%.
†Values are expressed Žmean " SD. as nmol mgy1 of urinary creatinine. Values in parentheses express percent of total. T-Cys, total cysteine; TF-Cys, total free cysteine; T-Hcy, total homocysteine; TF-Hcy, total free homocysteine; T-NAC, total N-acetylcysteine; TF-NAC, total free N-acetylcysteine; ‡not statistically significant with respect of corresponding basal values; U statistically Ž P- 0.01. significant from corresponding basal values. Day I, urinary levels of the day before infusion; Day II, urinary levels of the day of infusion; day III, urinary levels of the day after infusion.
Also, urinary free forms of Cys and Hcy increase on the day of NAC infusion, though to a lower extent with respect to the total corresponding amount, this indicates a reduction of percentage of Cys and Hcy excreted as the free form. Since none of the patients had significant proteinuria ŽTable I., the ‘free’ form of urine thiols can be identified in the ‘reduced’ form: thus in urine the difference between the total and free form reflects the ‘mixed disulphide’ amount. These data are consistent with a higher renal NAC-induced excretion of thiols as mixed disulphides Žprobably NAC᎐cysteine and NAC᎐homocysteine mixed disulphide.. Nevertheless, it has to be noticed that the absolute increase of Hcy and Cys excreted as the reduced form on the day of NAC infusion may also reflect a competition of NAC excess for tubular reabsorption sites w24x. In conclusion, a single NAC intravenous administration induces an efficient and rapid decrease of the main circulating plasma thiols ŽCys and Hcy.. This effect seems particularly relevant for Hcy and may be partly explained by displacement by NAC of thiols from their plasma binding sites Žplasma proteins. and by the formation, in large excess of plasma NAC, of NAC᎐Cys and NAC᎐Hcy mixed disulphides, which in turn undergoes a higher renal clearance, resulting in an increase of urinary excretion, likely as mixed disulphide forms. This approach may be of interest in decreasing plasma levels of atherogenic aminothiol homocysteine, particularly as an association therapy or as an
alternative approach in hyperhomocysteinaemic conditions poorly sensitive to vitamin administration w25, 26x. NAC may also have other important protective effects against Hcy toxicity. Its antioxidant capacity is well known, particularly against hydrogen peroxide w27x, an important intermediate in Hcy-induced toxicity w28x. Moreover, according to our data, NAC administration seems to decrease the plasma levels of the reduced form of Hcy, the one available to transformation in homocysteine᎐thiolactone, one of the most toxic Hcy metabolites w28x; from this point of view also, an increase of mixed-disulphide formation may represent a positive effect. As already mentioned, the effect of intravenous single infusion seems to be transient Žthe thiol levels tend to return to basal values on the day following the infusion.; this fact, together with the observed reduction of cysteine levels, is not a favourable effect: thus chronical administration studies, particularly about NAC oral administration, are needed before making conclusions about its effectiveness in the treatment of hyperhomocysteinaemic conditions.
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