- Email: [email protected]
Penicillamine
186
SEPARATION AND ANALYSIS
[33]
[33] P e n i c i l l a m i n e B y ELISAaETH M. WOLF-HEUSS
Introduction o-Penicillamine ( 2 - a m i n o - 3 - m e r c a p t o - 3 - m e t h y l b u t a n o i c acid) is a trifunctional thiol a m i n o acid. ~ It is widely used for the t r e a t m e n t o f a variety o f diseases including r h e u m a t o i d arthritis, 2 W i l s o n ' s disease, 3 cystinuria, 4 h e a v y metal poisoning, 5 chronic active hepatitis, 6 and p r i m a r y biliary cirrhosis. 7 T h e d e t e r m i n a t i o n o f o-penicillamine in biological fluids o f patients being treated with this drug is c o m p l i c a t e d by the fact that openicillamine o c c u r s in m a n y different f o r m s ; free thiol, internal disulfide, the m i x e d disulfide with cysteine, the metabolite S-methyl-D-penicillamine, and o-penicillamine b o u n d to p l a s m a proteins m a y all be present in b l o o d and urine samples. 8 A s s a y m e t h o d s e m p l o y i n g h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) , g a s - l i q u i d c h r o m a t o g r a p h y , amino acid analysis, colorimetry, and r a d i o i m m u n o a s s a y h a v e b e e n described. 9,j° Liquid C h r o m a t o g r a p h y D e t e r m i n a t i o n by E l e c t r o c h e m i c a l D e t e c t i o n Principle. D-Penicillamine and o t h e r sullhydryl-containing c o m p o u n d s are s e p a r a t e d on a c a t i o n - e x c h a n g e c o l u m n or r e v e r s e d - p h a s e H P L C c o l u m n and quantified by e l e c t r o c h e m i c a l detection. Saetre and R a b e n stein used a m e r c u r y - b a s e d e l e c t r o c h e m i c a l detector, ~,~2 which was W. M. W e i g e r t , H. O f f e r m a n n s , a n d P. S c h e r b e r i c h , Angew. Chem. 87, 374 (1975). 2 1. J. Jaffe, Arthritis Rheum. 8, 1064 (1965). 3 j . M. W a l s h e , Am. J. Med. 21, 487 (1956).
4 j. C. Crawhall, E. F. Scowen, and R. W. E. Watts, Br. Med. J. 1, 588 (1963). L. T. Zimmer and D. E. Carter, Life Sci. 23, 1025 (1978). R. B. Stern, S. P. Wilkinson, P. N. J. Howorth, and R. Williams, Gut 18, 19 (1977). 7 E. R. Dickson, C. R. Fleming, M. C. Geall, J. T. McCall, and A. H. Baggenstoss, Gastroenterology 72, 1049 (1977). 8 A. O. Muijsers, R. J. van de Stadt, A. M. A. Henrichs, and J. K. van der Korst, Clin. Chim. Acta 94, 173 (1979). 9 D. R. Lecavalier and J. C. Crawhall, J. Rheumatol. 8, Suppl. 7, 20 (1981). ~0j. C. Crawhall, Clin. Invest. Med. 7, 31 (1984). " R. Saetre and D. L. Rabenstein, Anal. Chem. 50, 276 (1978). t2 A. S. Russell, R. Saetre, P. Davis, and D. L. Rabenstein, J. Rheumatol. 6, 15 (1979). METHODS IN ENZYMOLOGY, VOL. 143
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[33]
PENICILLAMINE
187
slightly modified by Bergstrom et al., 13 and Carruthers et al. 14 used a commercially available gold-amalgam electrode. In the technique of Saetre and Rabenstein, blood samples are centrifuged to separate the plasma, and plasma proteins are precipitated with metaphosphoric acid prior to analysis. In order to determine both reduced and oxidized D-penicillamine in one analytical run, an aliquot of the protein-free plasma filtrate is electrolyzed for 10 min in an electrolysis cell to reduce D-penicillamine disulfides to free penicillamine. Protein-bound Dpenicillamine and S-methyl-D-penicillamine cannot be measured by this method. It is essential to deproteinate blood samples immediately upon collection so as to avoid the loss of free penicillamine by binding to human serum albumin. 15 Dithiothreitol has been used to reduce possible disulfides of D-penicillamine and protein-bound penicillamine for the determination of total penicillamine in plasma. 16Two independent assay methods were used for quantitating free penicillamine or penicillamine disulfide by direct injection of unreduced plasma filtrates using reversed-phase ion-pairing chromatography with electrochemical detection. A rapid and sensitive HPLC method, described by Kreuzig and Frank, 17 will be outlined here. It is essential to stabilize the samples immediately after collection by addition of EDTA because of the instability of D-penicillamine in solution against oxidation particularly at neutral pH and in the presence of heavy metals. A low sample pH is necessary. P r o c e d u r e . PREPARATION OF PLASMA SAMPLES. To ! ml of freshly drawn blood add 100 /~l of a 10% EDTA solution. After shaking, the mixture is centrifuged. Mix 1 volume of the supernatant fluid with 2 volumes of a solution prepared by dissolving 4.39 g of diammonium hydrogen citrate in about 900 ml of water, adding 100 g of metaphosphoric acid, and adjusting the volume to 1 liter with water. The precipitated proteins are removed by centrifugation. If the analysis is not performed immediately, the samples must be frozen with liquid nitrogen. PREPARATION OF URINE SAMPLES. To ] ml of fresh urine, add 100 pA of a 10% EDTA solution. Mix I volume of this sample with 2 volumes of the aqueous solution of metaphosphoric acid in diammonium hydrogen citrate described above. If turbidity results, the solution is centrifuged. C H R O M A T O G R A P H I C CONDITIONS. The HPLC separation is performed ~3 R. F. Bergstrom, D. R. Kay, and J. G. Wagner, J. Chromatogr. 222, 445 (1981). ~4 G. C a r r u t h e r s , M. Harth, D. F r e e m a n n , D. Weir, R. Rothwell, and M. Butler, Clin. Invest. Med. 7, 35 (1984). ~5 R. F. Bergstrom, D. R. Kay, and J. G. Wagner, Life Sci. 27, 189 (1980). ~6 M. A. A b o u n a s s i f and T. M. Jefferies, J. Pharm. Biomed. Anal. l, 65 (1983). 17 F. Kreuzig and J. Frank, J. Chromatogr. 218, 615 (1981).
188
SEPARATIONAND ANALYSIS
[33]
using a cation-exchange column (Nucleosil 5 SA 200/6/4, No. 715300 Machery-Nagel & Co, D~iren, FRG). The column is operated at a flow rate of 1.5 ml/min corresponding to a back pressure of 150 bars. The electrochemical detection unit consists of a flow-through cell (EA 1096/2, Metrohm, Switzerland) equipped with a three-electrode system (working electrode, gold, EA 286/3; reference electrode, silver-silver chloride with 3 M potassium chloride solution, EA 442; auxiliary electrode, glassy carbon, EA 286/1, Metrohm, Switzerland) and an amperometric detector (EA 611, Metrohm). The polarization voltage is adjusted to +800 mV. Plasma samples are detected at a signal sensitivity of 12.5 × 10 8 A, urine samples at a signal sensitivity of 12.5 × 10 7 A. In the case of plasma samples 20 mM diammonium hydrogen citrate is taken as eluent, whereas for urine samples l0 mM diammonium hydrogen citrate is used. The pH of the eluent is adjusted to 2.2 by means of phosphoric acid, followed by filtration through a 0.5-/~m Millipore filter and degassing prior to use. Standard solutions are prepared containing 1 and 5/~g of o-penicillamine, and 100/~g of EDTA per milliliter of eluent, for plasma and urine samples, respectively. The injection volume is 20 p3. After injection of five samples, a standard solution is injected. The retention time of D-penicillamine in plasma samples is 6 min with a detection limit of 50 ng/ml. When assaying urine samples under these chromatographic conditions, the retention time of n-penicillamine is 7.4 rain with a detection limit of 0.2/~g/ml. The coefficient of variation is 2.9% (n = 10). Specificity. The assay method is specific for reduced o-penicillamine.
Determination by Derivatization Principle. An HPLC method for the quantitative determination of Openicillamine in urine has been described using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) for postcolumn derivatization of free sulfhydryl groups, i~ Analytical methods for assaying D-penicillamine in plasma by selective precolumn derivatization of the sulfhydryl group are described. Separations are performed by reversed-phase liquid chromatography and fluorescence detection. Fluorescence derivatization prevents oxidation of D-penicillamine during analysis and enhances the sensitivity of the assay. 5-Dimethylaminonaphthalene-l-sulfonylaziridine19 and N-[p-(2-benzoxazolyl)phenyl]maleimide (BOPM) in 4- to 5-fold excess 2° have been used as derivatization reagents. ~ D. Beales, R. Finch, and A. E. M. M c L e a n , J. Chromatogr. 226, 498 (1981). ~9 E. P. L a n k m a y r , K. W. Budna, and K. Mfiller, J. Chromatogr. 222, 249 (1981). 2o j. O. Miners, 1. Fearnley, K. J. Smith, and D. J. Birkett, J. Chromatogr. (Biomed. Appl.) 275, 89 (1983).
[33]
PENICILLAMINE
189
Reagents and Standards. D-Penicillamine stock solution is prepared by dissolving D-penicillamine standard substance in 0.1% EDTA to give a final concentration of 5 mM. This solution is further diluted with 0.1% EDTA and with plasma to prepare calibration standards in the concentration range of 1 to 100 ttM. Procedure. STEP 1. PROTEIN PRECIPITATION. Immediately after collection, blood samples are transferred to 1.5-ml Eppendorf microtubes and centrifuged at 6500 g for 30 sec to obtain the plasma. An aliquot of 1 ml plasma is treated in a second Eppendorf microtube with 0.15 ml of 25% trichloroacetic acid. The samples are vortexed and cooled in an ice bath for 10 min in order to complete the plasma protein precipitation process. The proteins are separated by centrifugation at 6500 g for 2 min. STEP 2. DERIVATIZATIONWITH BOPM. Plasma supernatant, 0.5 ml, is transferred into a 5-ml glass tube and neutralized with 0.2 ml of 1% aqueous sodium hydroxide solution. The pH is adjusted to 5.0 by addition of 0.25 ml of a 0.5 M sodium citrate solution (titrated to pH 5.0 with perchloric acid). The derivatization reagent, 1 ml (1 mM BOPM in ethanol), is added. The mixture is incubated at 37° overnight. The penicillamineBOPM complex is stable for at least 24 hr. CHROMATOGRAPHIC CONDITIONS. The separation is done on a 10/zm reversed-phase /~Bondapak CIs column (30 cm × 3.9 mm i.d., Waters Assoc.). The mobile phase consists of methanol : 0.1 mM sodium acetate (48:52). A flow rate of 2.0 ml/min is maintained. Detection is with a Model 970 fluorescence detector (Spectra Physics). The excitation wavelength is set at 319 nm, and emission is measured using a 360 nm cut-off filter. The injection volume is 50/~1. Unknown concentrations of o-penicillamine in patient plasma are determined by comparison of the penicillamine peak heights with those of the calibration curve. The retention time of the penicillamine-BOPM derivative is 5.5 min. Fluorescence detection enables the quantitation of plasma penicillamine concentrations in the range of 0.25 to 500 ~M. Specificity. The method is specific for reduced D-penicillamine. The sulfhydryl-specific reagent BOPM does not react with the major metabolites of penicillamine, e.g., penicillamine disulsulfide or penicillaminecysteine disulfide. Disulfides may be determined as described above after Determination by Electrochemical Reduction. ~1,~7 Under the chromatographic conditions employed, BOPM complexes formed with cysteine and glutathione do not interfere.
Determination by Amino Acid Autoanalysis Principle. Quantitation of D-penicillamine in human plasma and urine by an amino acid autoanalyzer method has been described, s D-Penicil-
190
SEPARATION AND ANALYSIS
[33]
iamine and its disulfides are oxidized with performic acid to penicillaminic acid, which is separated from other ninhydrin-positive compounds as cysteic acid by anion-exchange chromatography. The possible metabolite, S-methyl-o-penicillamine,21 is oxidized by performic acid to a sulfone and separated by cation-exchange chromatography. Procedure. Freshly prepared performic acid, 2 ml (98% formic acid, 30% H202, 9 : 1 v/v), are added to 1 ml of serum or urine. The mixtures are incubated 1 hr at room temperature and 4 hr at 0°. HBr, 0.3 ml of a 48% solution, is added and centrifuged at 3000 g for 30 min. The supernatant liquid is brought to dryness and dissolved in 67 mM sodium citrate (pH 2.2) prior to analysis. Standards for calibration purposes are prepared by treating D-penicillamine and S-methyl-o-penicillamine standard substance in drug-free serum or urine. CHROMATOGRAPHIC CONDITIONS: SEPARATION OF" D-PENICILLAMINIC
ACID. Separation is done with a Beckmann Muitichrom M amino acid analyzer equipped with a 40 x 0.4 cm column of Aminex A-25 (Bio-Rad Laboratories). The column is regenerated with 0.2 M NaOH, briefly equilibrated with 0.8 M sodium citrate (pH 1.8), and equilibrated with 0.01 M acetic acid. Sample sizes of 50 to 150/zl are injected. Elution is performed at a column temperature of 65 ° with 10 mM acetic acid at a flow rate of 18 ml/hr. D-Penicillaminic acid is detected by postcolumn reaction with ninhydrin (10 ml/hr flow rate for ninhydrin). SEPARATION OF S - M E T H Y L - D - P E N I C I L L A M I N E SULFONE. m 40 X 0.4 cm cation-exchange column packed with Beckman resin M-902 is used. After regeneration with 0.2 M NaOH, the column is equilibrated with 67 mM sodium citrate (pH 2.2). Elution is performed with the same citrate buffer at a column temperature of 65° and at a flow rate of 25 ml/hr. Specificity. The method enables the determination of all forms of openicillamine. Protein-bound D-penicillamine is released by performic acid oxidation. The detection limit is 2/zM o-penicillamine in serum and urine samples. Assay by Colorimetry
Principle. A simple colorimetric procedure is based on derivatization of reduced D-penicillamine with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). 22 Procedure. Ten parts of blood sample and 1 part of Tris-EDTA (25 mM Tris, 100 mM EDTA, 120 mM NaCI, and 5 mM KCI, adjusted to pH 21 D. Perrett, W. Sneddon, and A. D. Stephens, Biochem. Pharmacol. 25,259 (1976). 22 j. Mann and P. D. Mitchell, J. Pharm. Pharmacol. 31, 420 (1979).
[34]
S-ADENOSYLMETHIONINE
AND
ITS SULFUR
METABOLITES
191
7.4) are mixed in an ice bath and centrifuged at 800 g for 10 min. An aliquot of I. l ml of plasma is mixed with 0. l ml 3 M HCI, frozen in a CO2methanol bath and lyophilized overnight. Ethanol, 1.5 ml, is added to the freeze-dried plasma sample. The suspension is sonicated for 5 to 10 sec and vortexed for 30 sec. After centrifugation at 800 g for l0 min, 1 ml of the supernatant fluid is added to 1 ml of 0.45 M Tris-HCl of pH 8.2 and mixed with 20 tzl of Ellman's reagent (10 mM in 0.1 M potassium phosphate at pH 7.0). Diethyl ether, 4 ml, is added, the tube is shaken for l0 min and centrifuged at 800 g for 10 min. The ethanol-ether layer is discarded, and the absorbance of the aqueous layer is measured at 412 nm. The calibration curve is obtained by adding known amounts of o-penicillamine standard substance to drug-free plasma and treating the samples as described. Specificity. The detection limit of o-penicillamine is 2/xg/ml. The calibration curve is linear up to 50 tzg/ml. Ellman's reagent has a limited range of application because it fails to distinguish between the drug and other thiols present in plasma samples. Penicillamine disulfides are not determined by this method.
[34] S-Adenosylmethionine a n d Its S u l f u r Metabolites By
RICHARD K. GORDON, GEORGE A. MIURA, TERESA ALONSO, and PETER K. CHIANG
Introduction Biological reactions in which S-adenosylmethionine (AdoMet) ~ is the methyl donor are key processes in many diverse cellular functions, such as transmethylation, transsulfuration, and polyamine biosynthesis. 2 Some of the key metabolites of AdoMet are S-adenosylhomocysteine (AdoHcy), decarboxylated S-adenosylmethionine (dcAdoMet), and methylthioadenosine (MeSAdo). The use of specific inhibitors of SThe following abbreviations are used: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; AdoCys, S-adenosylcysteine; Ade, adenine; dzAdo, 3-deazaadenosine: dzAdoHcy, S-3-deazaadenosylhomocysteine; dcAdoMet, decarboxylated S-adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine]; dcAdoHcy, decarboxylated Sadenosylhomocysteine IS-adenosyl-(5' )-3-thiopropylaminel; MeSAdo, 5'-deoxy-5'-melhylthioadenosine: lnoHcy, S-inosylhomocysteine. 2 E. Usdin, R. T. Borchardt, and C. R. Creveling, "Biochemistry of S-Adenosylmethionine and Related Compounds." Macmillan, London, 1982.
METHODS IN ENZYMOLOGY, VOL, 143
Copyright (~3 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
SEPARATION AND ANALYSIS
[33]
[33] P e n i c i l l a m i n e B y ELISAaETH M. WOLF-HEUSS
Introduction o-Penicillamine ( 2 - a m i n o - 3 - m e r c a p t o - 3 - m e t h y l b u t a n o i c acid) is a trifunctional thiol a m i n o acid. ~ It is widely used for the t r e a t m e n t o f a variety o f diseases including r h e u m a t o i d arthritis, 2 W i l s o n ' s disease, 3 cystinuria, 4 h e a v y metal poisoning, 5 chronic active hepatitis, 6 and p r i m a r y biliary cirrhosis. 7 T h e d e t e r m i n a t i o n o f o-penicillamine in biological fluids o f patients being treated with this drug is c o m p l i c a t e d by the fact that openicillamine o c c u r s in m a n y different f o r m s ; free thiol, internal disulfide, the m i x e d disulfide with cysteine, the metabolite S-methyl-D-penicillamine, and o-penicillamine b o u n d to p l a s m a proteins m a y all be present in b l o o d and urine samples. 8 A s s a y m e t h o d s e m p l o y i n g h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) , g a s - l i q u i d c h r o m a t o g r a p h y , amino acid analysis, colorimetry, and r a d i o i m m u n o a s s a y h a v e b e e n described. 9,j° Liquid C h r o m a t o g r a p h y D e t e r m i n a t i o n by E l e c t r o c h e m i c a l D e t e c t i o n Principle. D-Penicillamine and o t h e r sullhydryl-containing c o m p o u n d s are s e p a r a t e d on a c a t i o n - e x c h a n g e c o l u m n or r e v e r s e d - p h a s e H P L C c o l u m n and quantified by e l e c t r o c h e m i c a l detection. Saetre and R a b e n stein used a m e r c u r y - b a s e d e l e c t r o c h e m i c a l detector, ~,~2 which was W. M. W e i g e r t , H. O f f e r m a n n s , a n d P. S c h e r b e r i c h , Angew. Chem. 87, 374 (1975). 2 1. J. Jaffe, Arthritis Rheum. 8, 1064 (1965). 3 j . M. W a l s h e , Am. J. Med. 21, 487 (1956).
4 j. C. Crawhall, E. F. Scowen, and R. W. E. Watts, Br. Med. J. 1, 588 (1963). L. T. Zimmer and D. E. Carter, Life Sci. 23, 1025 (1978). R. B. Stern, S. P. Wilkinson, P. N. J. Howorth, and R. Williams, Gut 18, 19 (1977). 7 E. R. Dickson, C. R. Fleming, M. C. Geall, J. T. McCall, and A. H. Baggenstoss, Gastroenterology 72, 1049 (1977). 8 A. O. Muijsers, R. J. van de Stadt, A. M. A. Henrichs, and J. K. van der Korst, Clin. Chim. Acta 94, 173 (1979). 9 D. R. Lecavalier and J. C. Crawhall, J. Rheumatol. 8, Suppl. 7, 20 (1981). ~0j. C. Crawhall, Clin. Invest. Med. 7, 31 (1984). " R. Saetre and D. L. Rabenstein, Anal. Chem. 50, 276 (1978). t2 A. S. Russell, R. Saetre, P. Davis, and D. L. Rabenstein, J. Rheumatol. 6, 15 (1979). METHODS IN ENZYMOLOGY, VOL. 143
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[33]
PENICILLAMINE
187
slightly modified by Bergstrom et al., 13 and Carruthers et al. 14 used a commercially available gold-amalgam electrode. In the technique of Saetre and Rabenstein, blood samples are centrifuged to separate the plasma, and plasma proteins are precipitated with metaphosphoric acid prior to analysis. In order to determine both reduced and oxidized D-penicillamine in one analytical run, an aliquot of the protein-free plasma filtrate is electrolyzed for 10 min in an electrolysis cell to reduce D-penicillamine disulfides to free penicillamine. Protein-bound Dpenicillamine and S-methyl-D-penicillamine cannot be measured by this method. It is essential to deproteinate blood samples immediately upon collection so as to avoid the loss of free penicillamine by binding to human serum albumin. 15 Dithiothreitol has been used to reduce possible disulfides of D-penicillamine and protein-bound penicillamine for the determination of total penicillamine in plasma. 16Two independent assay methods were used for quantitating free penicillamine or penicillamine disulfide by direct injection of unreduced plasma filtrates using reversed-phase ion-pairing chromatography with electrochemical detection. A rapid and sensitive HPLC method, described by Kreuzig and Frank, 17 will be outlined here. It is essential to stabilize the samples immediately after collection by addition of EDTA because of the instability of D-penicillamine in solution against oxidation particularly at neutral pH and in the presence of heavy metals. A low sample pH is necessary. P r o c e d u r e . PREPARATION OF PLASMA SAMPLES. To ! ml of freshly drawn blood add 100 /~l of a 10% EDTA solution. After shaking, the mixture is centrifuged. Mix 1 volume of the supernatant fluid with 2 volumes of a solution prepared by dissolving 4.39 g of diammonium hydrogen citrate in about 900 ml of water, adding 100 g of metaphosphoric acid, and adjusting the volume to 1 liter with water. The precipitated proteins are removed by centrifugation. If the analysis is not performed immediately, the samples must be frozen with liquid nitrogen. PREPARATION OF URINE SAMPLES. To ] ml of fresh urine, add 100 pA of a 10% EDTA solution. Mix I volume of this sample with 2 volumes of the aqueous solution of metaphosphoric acid in diammonium hydrogen citrate described above. If turbidity results, the solution is centrifuged. C H R O M A T O G R A P H I C CONDITIONS. The HPLC separation is performed ~3 R. F. Bergstrom, D. R. Kay, and J. G. Wagner, J. Chromatogr. 222, 445 (1981). ~4 G. C a r r u t h e r s , M. Harth, D. F r e e m a n n , D. Weir, R. Rothwell, and M. Butler, Clin. Invest. Med. 7, 35 (1984). ~5 R. F. Bergstrom, D. R. Kay, and J. G. Wagner, Life Sci. 27, 189 (1980). ~6 M. A. A b o u n a s s i f and T. M. Jefferies, J. Pharm. Biomed. Anal. l, 65 (1983). 17 F. Kreuzig and J. Frank, J. Chromatogr. 218, 615 (1981).
188
SEPARATIONAND ANALYSIS
[33]
using a cation-exchange column (Nucleosil 5 SA 200/6/4, No. 715300 Machery-Nagel & Co, D~iren, FRG). The column is operated at a flow rate of 1.5 ml/min corresponding to a back pressure of 150 bars. The electrochemical detection unit consists of a flow-through cell (EA 1096/2, Metrohm, Switzerland) equipped with a three-electrode system (working electrode, gold, EA 286/3; reference electrode, silver-silver chloride with 3 M potassium chloride solution, EA 442; auxiliary electrode, glassy carbon, EA 286/1, Metrohm, Switzerland) and an amperometric detector (EA 611, Metrohm). The polarization voltage is adjusted to +800 mV. Plasma samples are detected at a signal sensitivity of 12.5 × 10 8 A, urine samples at a signal sensitivity of 12.5 × 10 7 A. In the case of plasma samples 20 mM diammonium hydrogen citrate is taken as eluent, whereas for urine samples l0 mM diammonium hydrogen citrate is used. The pH of the eluent is adjusted to 2.2 by means of phosphoric acid, followed by filtration through a 0.5-/~m Millipore filter and degassing prior to use. Standard solutions are prepared containing 1 and 5/~g of o-penicillamine, and 100/~g of EDTA per milliliter of eluent, for plasma and urine samples, respectively. The injection volume is 20 p3. After injection of five samples, a standard solution is injected. The retention time of D-penicillamine in plasma samples is 6 min with a detection limit of 50 ng/ml. When assaying urine samples under these chromatographic conditions, the retention time of n-penicillamine is 7.4 rain with a detection limit of 0.2/~g/ml. The coefficient of variation is 2.9% (n = 10). Specificity. The assay method is specific for reduced o-penicillamine.
Determination by Derivatization Principle. An HPLC method for the quantitative determination of Openicillamine in urine has been described using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) for postcolumn derivatization of free sulfhydryl groups, i~ Analytical methods for assaying D-penicillamine in plasma by selective precolumn derivatization of the sulfhydryl group are described. Separations are performed by reversed-phase liquid chromatography and fluorescence detection. Fluorescence derivatization prevents oxidation of D-penicillamine during analysis and enhances the sensitivity of the assay. 5-Dimethylaminonaphthalene-l-sulfonylaziridine19 and N-[p-(2-benzoxazolyl)phenyl]maleimide (BOPM) in 4- to 5-fold excess 2° have been used as derivatization reagents. ~ D. Beales, R. Finch, and A. E. M. M c L e a n , J. Chromatogr. 226, 498 (1981). ~9 E. P. L a n k m a y r , K. W. Budna, and K. Mfiller, J. Chromatogr. 222, 249 (1981). 2o j. O. Miners, 1. Fearnley, K. J. Smith, and D. J. Birkett, J. Chromatogr. (Biomed. Appl.) 275, 89 (1983).
[33]
PENICILLAMINE
189
Reagents and Standards. D-Penicillamine stock solution is prepared by dissolving D-penicillamine standard substance in 0.1% EDTA to give a final concentration of 5 mM. This solution is further diluted with 0.1% EDTA and with plasma to prepare calibration standards in the concentration range of 1 to 100 ttM. Procedure. STEP 1. PROTEIN PRECIPITATION. Immediately after collection, blood samples are transferred to 1.5-ml Eppendorf microtubes and centrifuged at 6500 g for 30 sec to obtain the plasma. An aliquot of 1 ml plasma is treated in a second Eppendorf microtube with 0.15 ml of 25% trichloroacetic acid. The samples are vortexed and cooled in an ice bath for 10 min in order to complete the plasma protein precipitation process. The proteins are separated by centrifugation at 6500 g for 2 min. STEP 2. DERIVATIZATIONWITH BOPM. Plasma supernatant, 0.5 ml, is transferred into a 5-ml glass tube and neutralized with 0.2 ml of 1% aqueous sodium hydroxide solution. The pH is adjusted to 5.0 by addition of 0.25 ml of a 0.5 M sodium citrate solution (titrated to pH 5.0 with perchloric acid). The derivatization reagent, 1 ml (1 mM BOPM in ethanol), is added. The mixture is incubated at 37° overnight. The penicillamineBOPM complex is stable for at least 24 hr. CHROMATOGRAPHIC CONDITIONS. The separation is done on a 10/zm reversed-phase /~Bondapak CIs column (30 cm × 3.9 mm i.d., Waters Assoc.). The mobile phase consists of methanol : 0.1 mM sodium acetate (48:52). A flow rate of 2.0 ml/min is maintained. Detection is with a Model 970 fluorescence detector (Spectra Physics). The excitation wavelength is set at 319 nm, and emission is measured using a 360 nm cut-off filter. The injection volume is 50/~1. Unknown concentrations of o-penicillamine in patient plasma are determined by comparison of the penicillamine peak heights with those of the calibration curve. The retention time of the penicillamine-BOPM derivative is 5.5 min. Fluorescence detection enables the quantitation of plasma penicillamine concentrations in the range of 0.25 to 500 ~M. Specificity. The method is specific for reduced D-penicillamine. The sulfhydryl-specific reagent BOPM does not react with the major metabolites of penicillamine, e.g., penicillamine disulsulfide or penicillaminecysteine disulfide. Disulfides may be determined as described above after Determination by Electrochemical Reduction. ~1,~7 Under the chromatographic conditions employed, BOPM complexes formed with cysteine and glutathione do not interfere.
Determination by Amino Acid Autoanalysis Principle. Quantitation of D-penicillamine in human plasma and urine by an amino acid autoanalyzer method has been described, s D-Penicil-
190
SEPARATION AND ANALYSIS
[33]
iamine and its disulfides are oxidized with performic acid to penicillaminic acid, which is separated from other ninhydrin-positive compounds as cysteic acid by anion-exchange chromatography. The possible metabolite, S-methyl-o-penicillamine,21 is oxidized by performic acid to a sulfone and separated by cation-exchange chromatography. Procedure. Freshly prepared performic acid, 2 ml (98% formic acid, 30% H202, 9 : 1 v/v), are added to 1 ml of serum or urine. The mixtures are incubated 1 hr at room temperature and 4 hr at 0°. HBr, 0.3 ml of a 48% solution, is added and centrifuged at 3000 g for 30 min. The supernatant liquid is brought to dryness and dissolved in 67 mM sodium citrate (pH 2.2) prior to analysis. Standards for calibration purposes are prepared by treating D-penicillamine and S-methyl-o-penicillamine standard substance in drug-free serum or urine. CHROMATOGRAPHIC CONDITIONS: SEPARATION OF" D-PENICILLAMINIC
ACID. Separation is done with a Beckmann Muitichrom M amino acid analyzer equipped with a 40 x 0.4 cm column of Aminex A-25 (Bio-Rad Laboratories). The column is regenerated with 0.2 M NaOH, briefly equilibrated with 0.8 M sodium citrate (pH 1.8), and equilibrated with 0.01 M acetic acid. Sample sizes of 50 to 150/zl are injected. Elution is performed at a column temperature of 65 ° with 10 mM acetic acid at a flow rate of 18 ml/hr. D-Penicillaminic acid is detected by postcolumn reaction with ninhydrin (10 ml/hr flow rate for ninhydrin). SEPARATION OF S - M E T H Y L - D - P E N I C I L L A M I N E SULFONE. m 40 X 0.4 cm cation-exchange column packed with Beckman resin M-902 is used. After regeneration with 0.2 M NaOH, the column is equilibrated with 67 mM sodium citrate (pH 2.2). Elution is performed with the same citrate buffer at a column temperature of 65° and at a flow rate of 25 ml/hr. Specificity. The method enables the determination of all forms of openicillamine. Protein-bound D-penicillamine is released by performic acid oxidation. The detection limit is 2/zM o-penicillamine in serum and urine samples. Assay by Colorimetry
Principle. A simple colorimetric procedure is based on derivatization of reduced D-penicillamine with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). 22 Procedure. Ten parts of blood sample and 1 part of Tris-EDTA (25 mM Tris, 100 mM EDTA, 120 mM NaCI, and 5 mM KCI, adjusted to pH 21 D. Perrett, W. Sneddon, and A. D. Stephens, Biochem. Pharmacol. 25,259 (1976). 22 j. Mann and P. D. Mitchell, J. Pharm. Pharmacol. 31, 420 (1979).
[34]
S-ADENOSYLMETHIONINE
AND
ITS SULFUR
METABOLITES
191
7.4) are mixed in an ice bath and centrifuged at 800 g for 10 min. An aliquot of I. l ml of plasma is mixed with 0. l ml 3 M HCI, frozen in a CO2methanol bath and lyophilized overnight. Ethanol, 1.5 ml, is added to the freeze-dried plasma sample. The suspension is sonicated for 5 to 10 sec and vortexed for 30 sec. After centrifugation at 800 g for l0 min, 1 ml of the supernatant fluid is added to 1 ml of 0.45 M Tris-HCl of pH 8.2 and mixed with 20 tzl of Ellman's reagent (10 mM in 0.1 M potassium phosphate at pH 7.0). Diethyl ether, 4 ml, is added, the tube is shaken for l0 min and centrifuged at 800 g for 10 min. The ethanol-ether layer is discarded, and the absorbance of the aqueous layer is measured at 412 nm. The calibration curve is obtained by adding known amounts of o-penicillamine standard substance to drug-free plasma and treating the samples as described. Specificity. The detection limit of o-penicillamine is 2/xg/ml. The calibration curve is linear up to 50 tzg/ml. Ellman's reagent has a limited range of application because it fails to distinguish between the drug and other thiols present in plasma samples. Penicillamine disulfides are not determined by this method.
[34] S-Adenosylmethionine a n d Its S u l f u r Metabolites By
RICHARD K. GORDON, GEORGE A. MIURA, TERESA ALONSO, and PETER K. CHIANG
Introduction Biological reactions in which S-adenosylmethionine (AdoMet) ~ is the methyl donor are key processes in many diverse cellular functions, such as transmethylation, transsulfuration, and polyamine biosynthesis. 2 Some of the key metabolites of AdoMet are S-adenosylhomocysteine (AdoHcy), decarboxylated S-adenosylmethionine (dcAdoMet), and methylthioadenosine (MeSAdo). The use of specific inhibitors of SThe following abbreviations are used: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; AdoCys, S-adenosylcysteine; Ade, adenine; dzAdo, 3-deazaadenosine: dzAdoHcy, S-3-deazaadenosylhomocysteine; dcAdoMet, decarboxylated S-adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine]; dcAdoHcy, decarboxylated Sadenosylhomocysteine IS-adenosyl-(5' )-3-thiopropylaminel; MeSAdo, 5'-deoxy-5'-melhylthioadenosine: lnoHcy, S-inosylhomocysteine. 2 E. Usdin, R. T. Borchardt, and C. R. Creveling, "Biochemistry of S-Adenosylmethionine and Related Compounds." Macmillan, London, 1982.
METHODS IN ENZYMOLOGY, VOL, 143
Copyright (~3 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.