High-Performance Liquid Chromatography and Laser Desorption

AC Research Anal. Chem. 1997, 69, 3855-3860

Accelerated Articles

High-Performance Liquid Chromatography and Laser Desorption/Ionization Mass Spectrometry of Retinyl Esters T. Wingerath,† D. Kirsch,‡ B. Spengler,‡ R. Kaufmann,‡,§ and W. Stahl*,†,§

Institut fu¨ r Physiologische Chemie I, Institut fu¨ r Lasermedizin, and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universita¨ t, Du¨ sseldorf, Postfach 101007, D-40001 Du¨ sseldorf, Germany

Laser desorption/ionization mass spectrometry (LDI-MS) at 337 nm laser wavelength was used to analyze retinol and several long-chain fatty acid esters of retinol. Employing this ionization technique helped to overcome the inherent problems resulting from thermal instability of retinyl esters which render this group of compounds rather difficult for standard ionization techniques. Mass spectra were recorded with a linear time-of-flight instrument in positive ion mode. Under these conditions, retinyl esters formed radical molecular ions (M•+) and in addition fragmented by elimination of the fatty acyl chain to uniformely form a peak at m/z ) 269 u. The elimiation of carbon dioxide was also observed in the spectra. The method is suitable to identify specific retinyl esters in complex mixtures of these compounds. A gradient reversed-phase high-performance liquid chromatography (HPLC) is described for the separation of retinol and 15 related fatty acid esters within 28 min. HPLC was applied to separate retinyl esters from rat liver extracts. The LDI mass spectrum of the collected HPLC fraction of rat liver extract showed the molecular parent ions of retinyl myristate, pentadecanoate, palmitoleate, palmitate, heptadecanoate, linoleate, oleate, stearate, and 3,4-didehydroretinyl palmitate. LDI-MS was found to be more appropriate than matrix-assisted laser desorption/ionization for the described analytical task.

Retinoids exert profound effects on biological processes such as vision, reproduction, cell growth, and differentiation1-3 and †

Institut fu ¨ r Physiologische Chemie I. Institut fu ¨ r Lasermedizin. § Biologisch-Medizinisches Forschungszentrum. (1) De Luca, L. M. FASEB J. 1991, 5, 2924-2933. (2) Sporn, M. B.; Roberts, A. B.; Goodman, D. S. The Retinoids: Biology, Chemistry and Medicine, 2nd ed.; Raven Press: New York, 1994. ‡

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received attention as therapeutics in cancer and certain types of skin diseases.4 The predominant forms of retinoids in biological samples are retinol and its esters with long-chain fatty acids5,6 (for structures, see Figure 1). Retinol and related fatty acid esters are characterized by strong UV absorption due to their conjugated system of double bonds. Therefore reversed-phase high-performance liquid chromatography (HPLC) coupled with UV/visible detection is commonly used for isolation, separation, and identification of retinyl esters.6-9 Limitations of HPLC separation and the similarity of absorption spectra, however, do not allow for unequivocal characterization. These disadvantages can be eliminated by employing mass spectrometry (MS), which provides both molecular weight information and characteristic fragment ion information helpful for structural elucidation.10-16 Various kinds of ionization techniques, including electron impact (EI),10-15 positive or negative chemical ionization (PCI, (3) Olson, J. A. In Modern Nutrition in Human Health and Disease, 8th ed.; Shils, M. E., Olson, J. A., Shike, M., Eds.; Lea and Febiger: Philadelphia, PA, 1994; p 287. (4) Bollag, W; Holdener, E. E. Ann. Oncol. 1992, 3, 513-526. (5) Futterman, S.; Andrews, J. S. J. Biol. Chem. 1964, 239, 4077-4080. (6) Ross, A. C. Anal. Biochem. 1981, 115, 324-330. (7) Bhat, P. V.; Lacroix, A. J. Chromatogr. 1983, 272, 269-278. (8) Furr, H. C.; Cooper, D. A.; Olson, J. A. J. Chromatogr. 1986, 378, 45-53. (9) Furr, H. C. Methods Enzymol. 1990, 89, 85-94. (10) Lin, R. L.; Waller, G. R.; E. D. Mitchell; Yang, K. S.; Nelson, E. C. Anal. Biochem. 1970, 35, 435-441. (11) Elliot, W. H.; Waller. G. R. In Biochemical applications of mass spectrometry; Waller, G. R., Ed.; Wiley: New York, 1972; p 499. (12) Reid, R.; Nelson, E. C.; Mitchell, E. D.; McGregor, M. L.; Waller, G. R.; John, K. V. Lipids 1973, 8, 558-565. (13) Winkler, P. C.; Perkins, D. D.; Williams, W. K.; Brower, R. F. Anal. Chem. 1988, 60, 489-493. (14) Clifford, A. J., Jones, A. D.; Furr, H. C. Methods Enzymol. 1990, 189, 94104. (15) Furr, H. C.; Clifford, A. J.; Jones, A. D. Methods Enzymol. 1992, 213, 281290. (16) van Breemen, R. B.; Huang, C. R. FASEB J. 1996, 10, 1098-1101.

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Table 1. Peak Identification of Retinyl Esters

peaka

identification

1 2 3 4 5 6+7

retinyl acetate retinyl caprate retinyl linolenate retinyl laurate retinyl arachidonate retinyl palmitoleate/ linoleate retinyl myristate retinyl pentadecanoate retinyl oleate retinyl palmitate retinyl heptadecanoate retinyl stearate retinyl arachinate retinyl behenate

8 9 10 11 12 13 14 15

no. of C atoms no. of (fatty acid) CdC

LDI-MS M•+ b (m/z)

[M - CO2 (44)]•+

2 10 18 12 20 16/18

0 0 3 0 4 1/2

328.2 440.4 546.5 468.4 572.5 522.5/548.5

284.2 396.3 502.5 424.4 528.5 478.5/504.5

14 15 18 16 17 18 20 22

0 0 1 0 0 0 0 0

496.5 510.5 550.5 524.5 538.5 552.5 580.5 608.5

452.5 466.4 506.5 480.5 494.5 508.5 536.5 564.5

a Peak numbers as indicated in Figure 2. b Monoisotopic molecular mass.

Figure 1. Structures of retinol, retinyl acetate, retinyl palmitate, retinyl palmitoleate, retinyl linoleate, and 3,4-didehydroretinyl palmitate (all-trans form).

Figure 3. UV spectra of retinol and three retinyl ester standards; retinol (s), retinyl pentadecanoate (- ‚), retinyl palmitate (‚‚‚), and retinyl arachidonate (- -).

Figure 2. HPLC chromatogram of a mixture of retinyl esters (50100 ng on column). For chromatographic conditions, see Materials and Methods; peak identification is given in Table 1.

NCI),17,18 laser desorption/ionization (LDI),19 and electrospray (ESI)16 mass spectrometry have been applied for analysis of underivatized retinol and related retinoids. Mass spectrometry of retinoids suffers particularly from their thermal instability. Electron impact mass spectra of retinoids show extensive fragmentation with a large number of uncharacteristic peaks.11 The molecular ion signal typically represents less than 5% of the ions (17) Napoli, J. L.; Pramanik, B. C.; Williams, J. B.; Dawson, M. I.; Hopps, P. D. J. Lipid Res. 1985, 26, 387-392. (18) Tzimas, G.; Sass, J. O.; Wittfoht, W.; Elmazar, M. M. A., Ehlers, K.; Nau, H. Drug Metab. Dispos. 1994, 22, 928-936. (19) McMahon, J. M. Anal. Biochem. 1985, 147, 535-545.

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produced in these studies.14 Chemical ionization of retinol often leads to quantitative dehydration.14 Similar difficulties have also been observed in HPLC-ESI mass spectrometry of retinol and retinyl acetate.16 Upon protonation, both molecules eliminated the functional group and uniformely formed a base peak at m/z ) 269 u with almost no protonated molecule remaining. LDI appeared to be most promising but has only been used for analysis of retinoic acid so far.19 In the present investigation, we report the first application of LDI mass spectrometry to the analysis of long-chain fatty acid esters of retinol. The suitability of the method to detect retinyl esters in biological material was tested using rat liver extracts. In mammals, up to 80% of the body’s total retinol is present in the liver, mainly located in stellate cells. Retinyl esters with various fatty acids represent the major storage forms. Stellate cells are able to control the storage and mobilization of retinol, thus ensuring constant plasma retinol levels in spite of normal fluctuations in daily vitamin A intake. The regulation of retinol uptake, storage, and remobilization is quite complex, and the role of retinyl esters in the whole process is only partially understood.20,21 With (20) Blomhoff, R.; Green, M. H.; Norum, K. R. Annu. Rev. Nutr. 1992, 12, 3757.

Figure 4. Laser desorption/ionization mass spectra of retinol and related retinyl esters. LDI mass spectra of retinol (m/z ) 286 u, A), retinyl acetate (m/z ) 328 u, B), retinyl palmitate (m/z ) 524 u, C), and retinyl palmitoleate (m/z ) 522 u) and linoleate (m/z ) 548 u, D) are shown.

the presented method, a tool is provided to investigate specific retinyl esters and follow their metabolism in biological systems. MATERIALS AND METHODS Reagents and Materials. all-trans-Retinol and all-trans-retinyl acetate were purchased from Sigma (Munich, Germany). alltrans-3,4-Didehydroretinol was a gift from Hoffmann-La Roche (Basel, CH). All solvents used were HPLC grade and were obtained from Merck (Darmstadt, Germany). Synthesis of Retinyl Esters. Retinyl esters of caprate (10: 0), laurate (12:0), myristate (14:0), pentadecanoate (15:0), palmitate (16:0), heptadecanoate (17:0), stearate (18:0), arachinate (20: 0), behenate (22:0), palmitoleate (16:1), oleate (18:1), linoleate (18: 2), linolenate (18:3), and arachidonate (20:4) were synthesized from retinol reacting either with fatty acyl chloride or fatty acid anhydride (Merck, Darmstadt, Germany) according to published methods.22,23 The reference compound of 3,4-didehydroretinyl palmitate (for structure, see Figure 1) was synthesized from 3,4didehydroretinol and palmitoyl chloride. 3,4-Didehydroretinol (10 µmol) was dissolved in 1 mL of dichloromethane (dried over 4 Å molecular sieves) and 15 µmol of fatty acyl chloride or fatty acid anhydride was added to the solution. The reaction was started with 10 µL (72 µmol) of triethylamine. The reagent tube was flushed with nitrogen and stirred for 1-2 h in the dark at 34-36 °C. The mixture was extracted with n-hexane (5 mL), and the hexane solution was washed two times with 4 mL of water. Phase separation was obtained by centrifugation (10 min at 4500 rpm). The organic phase was evaporated under a gentle stream of nitrogen, and the residue was purified by HPLC. Extraction of Tissues. Livers were obtained from untreated male Wistar rats and stored at -70 °C until analysis. Rat liver (∼1 g) was thawed and homogenized in buffer (2 mmol/L KH2PO4/K2HPO4, 0.7 mmol/L EDTA, and 1.4 mmol/L ascorbate, pH (21) Blomhoff, R. Vitamin A in health and disease; Marcel Dekker: New York, 1994. (22) Huang, H. S.; Goodman, D. W. S. J. Biol. Chem. 1965, 240, 2839-2844. (23) Lenzt, B. R.; Barenholz, Y.; Thompson, T. E. Chem. Phys. Lipids 1975, 15, 216-221.

Figure 5. Proposed fragmentation pathways of retinyl esters. LDI mass spectra of retinyl esters are characterized by (A) elimination of fatty acyl chain and (B) decarboxylation.

7.2). The mixture was extracted with n-hexane/dichloromethane (5:1) as previously described for human serum.24 High-Performance Liquid Chromatography. For HPLC, a Merck/Hitachi Model 655 A-12 ternary solvent delivery system equipped with a Merck/Hitachi Model L-4200 UV/visible detector and a Merck/Hitachi L-5000 LC controller (Merck, Darmstadt, Germany) was used. For spectrophotometric peak identification, a diode array detector (Model 168, Beckman, Munich, Germany) was used. The absorption spectra of retinol and retinyl esters were recorded between 230 and 600 nm. Separations were performed on a 5 µm Suplex pKb 100 column (250 mm × 4.6 mm) (Supelco, Bellefonte, PA) with a 20 mm guard column. Chromatographic Procedures. Retinyl esters purification was carried out by isocratic elution with solvent A, acetonitrile/ methanol/dichloromethane/n-hexane (88:4:4:4, v/v/v/v), or sol(24) Wingerath, T.; Stahl, W.; Sies, H. Arch. Biochem. Biophys. 1995, 324, 385390.

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Figure 6. Laser desorption/ionization mass spectrum of a synthetic mixture of retinyl esters. Radical molecular ions of retinol (m/z ) 286 u) and 15 retinyl esters are shown.

Figure 7. HPLC chromatogram of rat liver extract. Separation of major retinyl esters is achieved by gradient reversed-phase HPLC as described for retinyl ester standards. Peaks 1-8 in the chromatogram are tentatively assigned as retinyl linoleate/palmitoleate (1), myristate (2), pentadecanoate (4), oleate (5), palmitate (6), heptadecanoate (7), stearate (8), and 3,4-didehydroretinyl palmitate (3). The major ester in rat liver extract is retinyl palmitate.

vent B, acetonitrile/methanol/dichloromethane/n-hexane (70:10: 10:10, v/v/v/v). Chromatographic separation of synthetic retinyl ester mixtures and the esters extracted from rat liver was achieved by gradient HPLC. Isocratic elution with solvent A for 14 min at 1 mL/min was followed by a linear gradient to 100% B over a 2 3858

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min period; isocratic elution with this final solvent composition continued for 14 min at 1.5 mL/min. Detection was at 325 nm. Retinyl esters were quantified using retinyl palmitate as standard. Detection limit was ∼400 pmol of retinyl palmitate/mL. Sample Preparation for LDI-MS. Samples for LDI analysis were prepared by redissolving the HPLC fractions of standard retinyl esters in ∼50 µL of acetone. A 10 µL aliquot (∼80 pmol) of this solution was pipetted onto the surface of a slightly preheated sample holder, where the solvent evaporated within a few seconds. To avoid surface reactions, a gold target was used. LDI Mass Spectrometry. Mass spectrometrical analysis was carried out on a home-built linear time-of-flight (TOF) MS instrument. The technical details of this instrument have been described elsewhere.25 Its main features are summarized as follows: For desorption and ionization of retinol derivatives, a pulsed nitrogen laser (VSL 337 ND, Laser Science Inc., Cambridge, MA) with a wavelength of 337 nm was used. Ions were accelerated to a kinetic energy of 20 keV. After passing the fieldfree drift region of 110 cm, ions were detected by a 75 mm diameter microchannel plate (MCP) detector. The first plate of the detector was biased to ground potential. Carotenoids and their derivatives, which are structurally similar to retinyl esters, have been investigated successfully recently by employing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.26,27 Interferences between matrix ion signals and analyte fragment ion signals in MALDI spectra of retinyl esters, however, suggested (non-matrix-assisted) LDI mass spectrometry for these studies. The high spectral absorption, the relatively high volatility, and the sufficient ionizability of retinyl esters allowed us to obtain mass spectra by LDI that were similar in quality to those obtainable by MALDI. The clarity of LDI spectra was additionally enhanced by the fact that due to their spectral properties retinyl esters were favored in ionizability and analytical sensitivity over other compounds of the liver extract. (25) Spengler, B.; Kirsch, D.; Kaufmann, R. J. Phys. Chem. 1992, 96, 96789684. (26) Kaufmann, R.; Wingerath, T.; Kirsch, D.; Stahl, W.; Sies, H. Anal. Biochem. 1996, 238, 117-128. (27) Wingerath, T.; Kaufmann, R.; Kirsch, D.; Stahl, W.; Sies, H. J. Agric. Food Chem. 1996, 44, 2006-2013.

Figure 8. Laser desorption/ionization mass spectrum of rat liver extract. An abundant fragment ion at m/z ) 269 u is observed due to elimination of the fatty acyl chain. Radical molecular ions of retinyl myristate (m/z ) 496 u), pentadecanoate (m/z ) 510 u), palmitoleate (m/z ) 522 u), palmitate (m/z ) 524 u), heptadecanoate (m/z ) 538 u), linoleate (m/z ) 548 u), oleate (m/z ) 550 u), stearate (m/z ) 552 u), and 3,4didehydroretinyl palmitate (m/z ) 522 u) are recorded.

All mass spectrometrical investigations described in this paper were thus obtained from neat samples, not employing matrix assistance. LDI spectra are shown in the mass range above m/z ) 240 u. No structural information was obtained upon analysis of the mass range below m/z ) 240 u. RESULTS AND DISCUSSION A synthetic mixture of retinol and 15 esters of retinol (see selected structures in Figure 1) was chromatographed by HPLC. Figure 2 shows that with the gradient reversed-phase HPLC system a baseline separation for most of the retinyl esters was achieved within 28 min (for peak identification, see Table 1). Retention times of retinyl esters increased with increasing acyl chain length and with increasing degree of saturation.5 Retinyl acetate had a slightly shorter retention time than retinol. No baseline separation was achieved for peaks 3-5, retinyl linolenate (peak 3), retinyl laurate (peak 4), and retinyl arachidonate (peak 5). Coelution was found for retinyl linoleate and retinyl palmitoleate (peak 6 + 7), most likely due to similarities in the polarity of both compounds. Direct identification of the retinol derivatives by interpretation of the observed absorption spectra is rather difficult, due to insufficient differences in the spectra (Figure 3). In contrast to UV spectroscopy, unequivocal identification of retinol and its fatty acid esters can be achieved by LDI-MS, as demonstrated by the mass spectra of retinol and four retinyl esters (Figure 4). Using LDI-MS in the positive ion mode, abundant odd-electron (radical) molecular ions (M•+) at m/z ) 286 u are formed from retinol (Figure 4A). Minor fragment ions are observed at m/z ) 269 u and 255 u, resulting from loss of OH (-17 u, allyl cleavage) and by loss of CH2OH (-31 u), respectively. The peak at m/z ) 309 u corresponds to the sodium-attached molecule [M + Na]+. The mass spectrum of retinyl acetate is presented in Figure 4B. The base peak of the spectrum is the radical molecular ion at m/z ) 328 u. Additionally a loss of the acetate group (-59 u, m/z ) 269 u) and a decarboxylation (CO2

Table 2. Peak Identification of Retinyl Esters in Rat Liver Extract by LDI-MS LDI-MS M•+ b peaka

identification

(m/z)

[M - CO2 (44)]•+ (m/z)

1 1 2 3

retinyl linoleate retinyl palmitoleate retinyl myristate 3,4-didehydroretinyl palmitate retinyl pentadecanoate retinyl oleate retinyl palmitate retinyl heptadecanoate retinyl stearate

548.5 522.5 496.5 522.5

504.5 c 452.4 c

510.5 550.5 524.5 538.5 552.5

466.5 506.5 480.5 494.5 508.5

4 5 6 7 8

a Peak numbers as indicated in Figure 7. b Monoisotopic molecular mass. c Not observed.

-44 u, m/z ) 284 u) is obtained. Formation of the deacetylated ion can be attributed to an allyl cleavage (see Figure 5A), whereas the decarboxylated ion is formed through a cyclic transition state.28,29 The proposed mechanism of the decarboxylation reaction is described in Figure 5B. The loss of CO2 has not been observed with other ionization techniques. Spectra of retinyl acetate recorded with other ionization techniques show no, or only a minor, molecular ion peak.13,15,16 In analogy to retinyl acetate and retinol, allyl cleavage of the fatty acyl group (-255 u) is observed for retinyl palmitate, expressed as the base peak at m/z ) 269 u in Figure 4C. The radical molecular ion is observed at m/z ) 524 u. Loss of CO2 from the molecular ion (-44 u) leads to an ion signal at m/z ) 480 u in analogy to retinyl acetate. Coeluting retinyl esters can be readily distinguished and identified by their molecular weight in the LDI mass spectrum as demonstrated for retinyl palmitoleate and linoleate (Figure 4D). (28) Bowie, J. H.; Williams, D. H.; Madsen, P.; Schrool, G.; Laesson, S.-O. Tetrahedron 1967, 23, 305-320. (29) Fischer, M., Djerassi, C. Chem. Ber. 1966, 99, 750-764.

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LDI-MS analysis of peak 6 + 7 shows the molecular parent ions (M•+) of retinyl palmitoleate at m/z ) 522 u and retinyl linoleate at m/z ) 548 u. The base peak at m/z ) 269 u is formed by elimination of the fatty acyl chains of both retinyl esters. Ions at m/z ) 478 u and 504 u result from the loss of CO2 from both parent ions. Successful analysis of complex mixtures of retinyl esters from biological samples requires not only the detectability of individual target components. Another prerequisite is that identification and detection efficiency is maintained in complex mixtures where physicochemical interactions might cause suppression of analyte signals or reduction of analytically required mass resolving power. In order to test the capability of the method to analyze complex mixtures of retinyl esters, a synthetic mixture of retinol and 15 retinyl esters was analyzed by LDI-MS (Figure 6). The base peak of the spectrum at m/z ) 269 u corresponds to the common retinyl residue and thus does not contain information on individual components. The enlarged mass range from 390 to 620 mass units exhibits the radical molecular ions of retinyl caprate (m/z ) 440 u), laurate (m/z ) 468 u), myristate (m/z ) 496 u), pentadecanoate (m/z ) 510 u), palmitoleate (m/z ) 522 u), palmitate (m/z ) 524 u), heptadecanoate (m/z ) 538 u), linolenate (m/z ) 546 u), linoleate (m/z ) 548 u), oleate (m/z ) 550 u), stearate (m/z ) 552 u), arachidonate (m/z ) 572 u), arachinate (m/z ) 580 u), and behenate (m/z ) 608 u). As expected from single-component analyses, most of the peaks are accompanied by satellite peaks [M - 44]•+ due to loss of CO2. The spectrum proves that mass resolution of the linear TOF mass spectrometer was high enough to distinguish between the most delicate retinyl esters of linolenate (m/z ) 546 u), linoleate (m/z ) 548 u), oleate (m/z ) 550 u), and stearate (m/z ) 552 u). HPLC and LDI-MS was applied to determine the retinyl ester pattern in rat liver extracts. Figure 7 shows the HPLC chromatogram of rat liver extract. Peaks 1-8 are tentatively assigned by comparison of HPLC retention times with the reference compounds as retinyl linoleate/palmitoleate (1), myristate (2), retinyl pentadecanoate (4), oleate (5), palmitate (6), heptadecanoate (7), and stearate (8). Peak 3 is tentatively assigned as 3,4-didehydroretinyl palmitate by HPLC retention times and its absorbance properties. An additional double bond in the 3,4-position of the β ring extends conjugation of the polyene chain and increases λmax from 325 to 350 nm. For LDI analyses of retinyl esters in rat liver extract, samples are prepurified by HPLC in order to separate the analytes from other lipophilic compounds (e.g., phospholipids, mono-and diglycerides, and fatty acids). The HPLC eluate collected between

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10 and 23 min was used for LDI-MS. The LDI spectrum is shown in Figure 8. As expected from the test mixture analysis, the ion signal from the common retinyl residue at m/z ) 269 u is the base peak again. The enlarged mass range from 440 to 570 mass units shows the molecular parent ions of eight retinyl esters as listed in Table 2. Retinyl palmitoleate and 3,4-didehydroretinyl palmitate have identical molecular masses. For further identification, peaks 1 and 3 (see Figure 7) were isolated and analyzed by LDI. The LDI spectrum of peak 1 showed a major peak at m/z ) 548 u assigned to retinyl linoleate and a minor peak at m/z ) 522 u assigned to retinyl palmitoleate. 3,4-Didehydroretinyl palmitate in peak 3 was identified by the molecular parent ion at m/z ) 522 u and the characteristic 3,4-didehydroretinyl residue at m/z ) 267 u. The specific fragment ion of 3,4-didehydroretinyl palmitate at m/z ) 267 u was not observed in the mass spectrum shown in Figure 8. This is likely due to the low concentration of this 3,4-didehydroretinyl ester in rat liver extract. As before, most of the molecular parent ion signals are accompanied by [M 44]•+ satellites due to loss of CO2. CONCLUSIONS Our studies demonstrate that LDI mass spectrometry in combination with HPLC is a suitable technique to analyze retinyl esters extracted from biological samples. Upon laser desorption/ ionization, these compounds form abundant radical molecular ions and fragment by elimination of the fatty acyl chain as well as by decarboxylation. The method is suitable to identify specific retinyl esters in complex mixtures. The efficiency of LDI-MS for biological analyses is shown for the case of imaging the retinyl ester pattern in rat liver extract. The identification and quantification of specific retinyl esters in biological samples will contribute to an understanding of the complex regulatory system underlying vitamin A homeostasis. ACKNOWLEDGMENT We gratefully acknowledge the helpful discussions with Prof. Dr. H. Sies, Institut fu¨r Physiologische Chemie I, University of Du¨sseldorf. The present study was supported by the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (Bonn, Germany). Received for review May 7, 1997. 1997.X

Accepted July 31,

AC970463W X

Abstract published in Advance ACS Abstracts, September 1, 1997.