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Formation of Cu or Fe thiobarbiturate complexes interfere with the determination of malondialdehyde
Journal of Inorganic Biochemistry 72 (1998) 217±225
Formation of Cu or Fe thiobarbiturate complexes interfere with the determination of malondialdehyde Lily Zhou, John R.J. Sorenson
*
Division of Medicinal Chemistry, College of Pharmacy, University of Arkansas for Medical Sciences Campus, Little Rock, Arkansas 72205, USA Received 16 July 1998; received in revised form 6 October 1998; accepted 22 October 1998
Abstract This research was conducted to determine whether or not Cu(II) and Fe(III) form TBA complexes that absorb and ¯uoresce at the same wavelengths as the bis-TBA-MDA adduct and support a false conclusion that Cu(II) and Fe(III) cause lipid and other tissue component oxidations as evidenced by the apparent formation of the bis-TBA-MBA adduct. Additions of Cu(II) or Fe(III) to Na TBA gave concentration related increases in absorbance at 532 nm, the wavelength of maximum absorbance of the bis-TBAMDA adduct. These absorbance spectra demonstrate that the addition of aqueous solutions of Na TBA to systems containing added Cu(II) and/or Fe(III) and their butanol extracts will support the false conclusion that Cu(II) and/or Fe(III) caused lipid and other tissue component oxidations. Fluorescence at 553 nm, the maximum for ¯uorescence of the bis-TBA-MDA adduct, increased as the concentration of Cu(II) increased but decreased with the addition of Fe(III). Fluorescence obtained following the addition of Cu(II) to Na TBA will support the false conclusion that Cu(II) causes lipid and other tissue component oxidations while the lack of ¯uorescence following the addition of Fe(III) will not support this false conclusion. These studies show that the presence of Cu(II) or Fe(III) in TBA assay systems lead to the specious interpretation that oxidations occurred due to the formation of HO´ radical via Cu(II)- or Fe(III)-mediated Fenton chemistry. Ó 1998 Elsevier Science Inc. All rights reserved. Keywords: Copper; Iron; Thiobarbituric acid; Absorbance; Fluorescence; Malondialdehyde
1. Introduction Unsaturated lipid [1±40], lipoprotein [27,30,35,37, 38,41±50], deoxyribose or DNA [15±18,24,25,31,35,50± 60], protein [18,25,27,30,31,61±63], carbohydrate [51,56], bile pigment [53], lens crystallins [61,64], catecholamines [34], and mylein [30] oxidative degradations are suggested to have a major pathological role in many disease states including arthritidies [15±17,19,53,56,62], atherosclerosis [27,33,35,37,40,41,43,45,47±49], central neuronal cell death [31,39], hemolysis [8,28,65], thrombosis [43], myocardial infarction [41,43,44], aceruloplasminemia [39], renal insuciency [32], diabetes [32,33], cardiovascular disease [27,37,43,44], peripheral vascular disease [37], pancreatitis [32], liver cirrhosis [32,63], cancer [32], angioplasty injury [48], radiation injury [7,8,18,50,56], ischemia-reperfusion injury [31,33,64], as well as Parkinson's [30,31,34], Alzheimer's [30,31], Menkes' [63], and Wilson's disease [15,17,33,39]. *
Corresponding Author. E-mail: [email protected]
These degradations and disease states are often modeled as in vitro biological systems by addition of hydrogen peroxide (H2 O2 ), vitamin C, or thiols and Fe and/or Cu and sometimes heating up to 100°C. These conditions are hypothesized to lead to the formation of an aggressive oxidant most frequently suggested to be hydroxyl radical (HO´ ) formed via Fenton chemistry [66]. Lower oxidation states of Cu(I) or Fe(II) in tissues are suggested to cause homolytic cleavage of hydrogen peroxide to yield HO´ ; Fe(II) or Cu(I) + H2 O2 ® Fe(III) or Cu (II) + HO´ OHÿ . The assumed formation of HO´ is then suggested to cause the formation of malondialdehyde (MDA), as shown in Scheme 1, which is then assumed to react with thiobarbituric acid (TBA) to account for the increase in measured absorbance at 532 nm and/or ¯uorescence at 553 nm. Perusal of the literature [1±65] revealed that nearly all experimental paradigms intended to produce HO´ mediated destruction involved the addition of something of the order of 10ÿ6 to 10ÿ3 M ionically bonded Cu(II) or Fe(III) to modeled biological systems. The
0162-0134/98/$ ± see front matter Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 1 0 0 8 3 - 1
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L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
Scheme 1.
highest concentration found was 0.3 M [48]. Since calculated concentrations of ionically bonded Cu and Fe in plasma are of the order of 10ÿ18 and 10ÿ23 M [67], respectively, and are too small to be measured with any exiting analytical method, additions of much larger amounts of ionically bonded Cu or Fe seem to lack relevance. Amounts of ionically bonded Cu and Fe in solid tissues are likely to be still lower due to a greater number of bonding sites. The remaining Cu and Fe found in all tissues exhists as stable complexes including Cu- and Fe-dependent enzymes, and these coordinate-covalently bonded forms of Cu and Fe do not represent ionically-bonded redox-active forms of these metalloelements that are likely to be serious threats to cells as HO generators. As a result, it was questioned as to whether or not additions of ionically bonded forms of Cu and Fe led to indiscriminate bonding of these metalloelements to components of these biological systems and had a role in causing the reported system de®cits that were interpreted as oxidative degradations. Another curious observation was the use of concentrations of H2 O2 ranging up to 33.3 M when the estimated concentration of H2 O2 in normal cells is of the order of 10ÿ8 M [68]. It was questioned as to whether or not these high concentrations were relevant since it is unlikely that cells containing much more than 10ÿ8 M H2 O2 would remain viable. The need for the use of large concentrations of H2 O2 in these experiments may arise from the fact that both Cu(II) and Fe(III) have catalasemimetic activity [69], and can convert lower concentrations of hydrogen peroxide to triplet state oxygen and water at rates of the order of 109 molÿ1 sÿ1 , and Cu(I) and Cu(II) as well as Fe(II) and Fe(III) are excellent HO´ scavengers with rates of reduction of hydroxyl radical, HO´ ® HOÿ , of the order of 1010 molÿ1 sÿ1 [70] with oxidation of these metalloelements to the next
higher oxidation state: Cu(II) and Cu(III) or Fe(III) and Fe(IV). Authentic HO´ generation is known to lead to the formation of MDA [2,23]. The TBA assay has been widely used as a test for MDA produced by lipid and other tissue component oxidations in modeled biological systems. This assay is based on the reaction of TBA with the bifunctional MDA to yield a bis-TBA-MDA adduct [2,23]. The resulting conjugated aromatic chromophore shown in Scheme 2 has a characteristic absorbance maximum at 532 nm and ¯uorescence maximum at 553 nm [71]. In the light of the above, it occurred to us that additions of Cu and Fe to modeled biological systems may also produce an absorbance at 532 nm and an emission at 553 nm following the addition of TBA due to the formation of Cu or Fe complexes of TBA, which may be falsely interpreted as tissue component oxidation by Cu or Fe based upon the TBA assay. Unfortunately these TBA assay results reported by many are not controlled for the addition of Cu or Fe as they may eect the TBA assay by increasing the observed absorbance due to formation of Cu or Fe complexes of TBA: Cu(II)(thiobarbiturate)2 or Fe(III)-(thiobarbiturate)3 , as shown in Scheme 3, and/or the oxidation of TBA to its disul®de by Cu(II) or Fe(III) and the formation of Cu(II) or Fe(III) complexes of the disul®de, as shown below in Scheme 4, where n is the number of TBA ligands and nI is the oxidation state of Cu(I or II) or Fe(II or III). To examine the possibility that ionically bonded Cu and Fe compounds form these complexes and/or TBA disul®de and cause a positive interference with the determination of the bis-MDA-TBA adduct, a product of lipid and other tissue component oxidations produced by HO,both absorbance and ¯uorescence spectra were obtained for mixtures of ionically bonded Cu and Fe compounds and TBA. 2. Materials and methods Cupric Chloride, [Cu(II)C12 ] (Spectrum Chemical Corp, A. C. S. Reagent), Ferric Chloride, [Fe(III)C13 ] (Mallinckrodt Chemical Works, Analytical Reagent), Ferric Sulfate, [Fe(III)2 (SO4 )3 ] (Alfa Products, Reagent), Sodium Hydroxide, (NaOH) (Aldrich Chemical Company, A. C. S. Reagent), Thiobarbituric Acid,
Scheme 2.
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
219
Scheme 3.
Scheme 4.
(TBA) (Sigma Chemical Co, 98% pure) were used without further puri®cation. Butanol or deionized water were used to prepare solutions. An IEC HN-S II centrifuge was used to centrifuge turbid solutions. One centimeter quartz cuvettes were used for spectrophotometric and ¯uorescence measurements. A HewlettPackard 8452a Diode Array Spectrophotometer with a Vectna E5 Computer and Printer was used to measure and record absorbance. A Perkin Elmer Luminescence Spectrometer was used to measure ¯uorescence from 200 to 800 nm. All glassware (beakers, Erlenmeyer ¯asks, volumetric ¯asks, graduated cylinders, medium porosity sintered fritted glass ®lter funnels, and suction ¯asks) were thoroughly cleaned with Citronox (Alconox Inc.), Acetone, or 10% Hydrochloric Acid (HCl). Other equipment including: spatulas, test tubes, Pasteur pipettes, and disposable tips for hand held pipettes were metal free. To prepare 100 ml of a 50 mM NaOH solution, 0.2 gm (5 mmol) of NaOH was dissolved in 100 ml of deionized H2 O. To prepare 100 ml of a 14.3 mM NaOH solution, 0.057 gm (1.43 mmol) of NaOH was dissolved in 100 ml of deionized water. To prepare 100 ml of a 70 mM TBA solution, 1.008 gm (7 mmol) of TBA was dissolved in 100 ml of 50 mM NaOH solution and ®ltered through a sintered glass funnel. To prepare 100 ml of a 7 mM solution of Cu(II)Cl2 (H2 O)2 , 0.229 gm (0.7 mmol) was dissolved in 100 ml of deionized water. To prepare 100 ml of a 35 mM solution of cupric chloride, 0.597 gm (3.5 mmol) of Cu(II)C12 (H2 O)2 was dissolved in 100 ml of deionized H2 O. To prepare 100 ml of a 23 mM solution of Fe(III)C13 , 0.622 gm (2.3 mmol) of Fe(III)C13 (H2 O)6 was dissolved in 100 ml of deionized H2 O. To prepare 100 ml of 4 mM solution of Fe(III)Cl3 , 0.1081 gm (0.4 mmol of Fe(III)Cl2 (H2 O)6 was dissolved in 100 ml of deionized water. To prepare
100 ml of a 23 mM solution of Fe(III)2 (SO4 )3 , 1.044 gm (2.3 mmol) of Fe(III)2 (SO4 )3 (H2 O)3 was dissolved in 100 ml of deionized H2 O and ®ltered through a sintered glass funnel. To obtain spectra for mixtures of Cu(II)C12 or Fe(III)2 (SO4 )3 [or Fe(III)C13 ] and TBA, absorbance and ¯uorescence were set at zero with a solution of 0.15 ml of 50 mM NaOH in 2.5 ml of deionized water. Fluorescence measurements were done in triplicate. The amount of TBA in these solutions was held constant and the amount of Cu(II)C12 or Fe(III)Cl 3 or Fe(III)2 (SO4 )3 added was varied as shown in Tables 1 and 2 respectively. A volume of deionized H2 O was added to the cuvette prior to the addition of TBA and either Cu(II)C12 or Fe(III)Cl3 or Fe(III)2 (SO4 )3 with thorough mixing to maintain a constant volume in the cuvette. The following procedures were used to obtain spectra of butanol extracted chromophore produced with the mixture of Cu(II) or Fe(III) and NaTBA solutions. 2 ml of 70 mM TBA in 50 mM NaOH and 2 ml of 35 mM Cu(II)C12 were pipetted into a metal-free polypropylene culture tube. Five ml of butanol was then added and the Table 1 Volumes and ®nal concentrations of TBA and Cu(II)Cl2 used to prepare solutions for absorbance and ¯uorescence measurements
1. 2. 3. 4. 5.
TBA (®nal concentration)
Cu(II)C12 (®nal concentration)
Deionized water
0.30 0.30 0.30 0.30 0.30
0.00 0.15 0.20 0.25 0.30
2.70 2.55 2.50 2.45 2.40
ml ml ml ml ml
(7 (7 (7 (7 (7
mM) mM) mM) mM) mM)
ml ml ml ml ml
(0.00 (1.75 (2.33 (2.92 (3.50
mM) mM) mM) mM) mM)
ml ml ml ml ml
220
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
Table 2 Volumes and ®nal concentrations of TBA and Fe(III)Cl2 or Fe(III)2 (SO4 )3 used to prepare solutions for absorbance and ¯uorescence measurements
1. 2. 3. 4. 5.
TBA (®nal concentration)
Fe(III)Cl3 or Fe(III)2 (SO4 )3 (®nal concentration)
Deionized H2 O
1.50 1.50 1.50 1.50 1.50
0.03 ml (0.23 mM) 0.06 ml (0.46 mM) 0.125 ml (0.96 mM) 0.25ml (1.92 mM) 0.50 ml (3.83 mM)
1.47 ml 1.44 ml 1.375 ml 1.25 ml 1.00 ml
ml ml ml ml ml
(35 (35 (35 (35 (35
mM) mM) mM) mM) mM)
mixture stirred with a vortex mixer for 5 minutes. The same procedure was repeated using 2 ml of 70 mM TBA in 50 mM NaOH, 2 ml of 23 mM Fe(III)C13 , and 5 ml of butanol. The butanol layer was removed from each mixture using metal-free disposable Pasteur pipettes and placed in two separate polypropylene culture tubes. Ultraviolet±Visible spectra were obtained for each butanol solution after setting the absorbance at zero using 3 ml of butanol. To examine stoichiometries of the reaction of Cu(II)Cl2 and Fe(III)Cl3 with TBA, concentrations of Cu(II)Cl2 and Fe(III)Cl3 were held constant and the concentration of NaTBA added was varied as shown in Tables 3 and 4, respectively. All spectral measurements were performed in triplicate. To examine the in¯uence of heating on the appearance of the chromophore, solutions represented in Tables 3 and 4 were heated at 100°C for 5 min and spectra recorded. This heating procedure caused precipitate formation in some solutions. When a precipitate formed the solution was centrifuged for 10 min at about 2000 revolutions per minute and a spectrum obtained for the supernatant. All determinations for the mixture of Cu(II)Cl2 or Fe(III)Cl3 with NaTBA were performed on the same day to avoid time-dependent variations. All measurements were done in triplicate.
Table 4 Volumes and concentrations of Fe(III)Cl3 and TBA solutions used to examine the stiochiometry of chromophore formation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
TBA (®nal concentration)
Fe(III)Cl3 (®nal concentration)
Deionized water
1.50 1.35 1.20 1.05 0.90 0.75 0.60 0.45 0.30 0.15 0.00
1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50
0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50
ml ml ml ml ml ml ml ml ml ml ml
(10 mM) (9 mM) (8 mM) (7 mM) (6 mM) (5 mM) (4 mM) (3 mM) (2 mM) (1 mM) (0 mM)
ml ml ml ml ml ml ml ml ml ml ml
(2 (2 (2 (2 (2 (2 (2 (2 (2 (2 (2
mM) mM) mM) mM) mM) mM) mM) mM) mM) mM) mM)
ml ml ml ml ml ml ml ml ml ml ml
3. Results Fig. 1 shows the increase in absorbance at 532 nm, which would be falsely interpreted as increased MDA formation, when increasing concentrations of Cu(II)C12 ranging from 0.00 to 3.50 mM were added to a 7 mM solution of NaTBA. There was a concentration related increase in absorbance and intensity of greenish-yellow color when concentrations of 2.3 mM Cu(II)Cl2 and larger were added to 7 mM NaTBA. An increase in the concentration of Cu(II)Cl2 above 3.5 mM resulted in the formation of a greenish-yellow precipitate, suggesting the formation of a mixture of a Cu(I) thiol and Cu(II) oxygen-nitrogen chelates of TBA or TBA disul®de. The increase in absorbance at 532 nm and intensity of red-wine color, consistent with the false interpretation that MDA formed, when increasing concentrations of
Table 3 Volumes and concentrations of Cu(II)Cl2 and TBA solutions used to examine the stoichiometry of chromophore formation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
TBA (®nal concentration)
Cu(II)Cl2 (®nal concentration)
Deionized water
1.50 1.35 1.20 1.05 0.90 0.75 0.60 0.45 0.30 0.15 0.00
1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50
0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50
ml ml ml ml ml ml ml ml ml ml ml
(10 mM) (9 mM) (8 mM) (7 mM) (6 mM) (5 mM) (4 mM) (3 mM) (2 mM) (1 mM) (0 mM)
ml ml ml ml ml ml ml ml ml ml ml
(3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5
mM) mM) mM) mM) mM) mM) mM) mM) mM) mM) mM)
ml ml ml ml ml ml ml ml ml ml ml
Fig. 1. Increase in absorbance at 532 nm for addition of Fe(III)Cl3 to 35 mN NaTBA or Cu(II)Cl2 to 7 mM NaTBA.
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
221
Fig. 3. Increase in ¯ourescence at 553 nm for addition of Cu(II)Cl2 to 7mM NaTBA or decrease in ¯oursence at 553 nm for addition of Fe(III)2 (SO4 )3 to 35 mM NaTBA.
Fig. 2. Time dependent incease in abosorbance at 532 nm for a butanol extract of a 5.11 mM Fe(III)Cl3 -15.56 mM NaTBA solution or a 7.78 mM Cu(II)Cl2 -15.56 mM NaTBA solution.
Fe(III)Cl3 ranging from 0.00 to 3.83 mM were added to a 35 mM solution of NaTBA is shown in Fig. 1. No precipitate formed when these concentrations of Fe(III)Cl3 were added to 35 mM NaTBA, which was ®ve times as concentrated as the NaTBA solution used for additions of Cu(II)Cl2 . Ultraviolet±Visible spectrophotometric measurements were also made for a butanol extract of the mixture of 7.78 mM Cu(II)Cl2 and 15.56 mM NaTBA and the mixture of 5.11 mM Fe(III)Cl3 and 15.56 mM NaTBA. Following extraction of these mixtures it was noticed that absorbances of these extracts changed over a period of 6 h. The absorbance at 532 nm increased as shown in Fig. 2 for the butanol extract of the Cu(II)Cl2 ± NaTBA solution through the period of this experiment while the 532 nm absorbance for the butanol extract of the Fe(III)Cl3 ±NaTBA solution decreased over the ®rst hour it did not change signi®cantly over the next 5 h (Fig. 2). The observed increase in absorbance for both butanol extracts would also be falsely interpreted as being due to the formation of MDA. The linear increase in ¯uorescence at 553 nm observed when increasing concentrations of Cu(II)Cl2 ranging from 0.00 to 3.50 mM were added to a 7 mM solution of NaTBA is shown in Fig. 3. This increase in ¯uorescence, would also lead to the false interpretation that MDA had been formed. Fig. 3 shows the ¯uorescence observed at 553 nm when increasing concentrations of Fe(III)2 (SO4 )3 ranging from 0.00 to 3.83 mM were added to a 35 mM solution of NaTBA. The ¯uorescence observed following the addition of 0.23 mM Fe(III)2 (SO4 )3 , 0.46 mM Fe(III), which was less than the ¯uorescence observed for the 35 mM solution of NaT-
BA, 0.07 ergs sÿ1 , still is consistent with the false notion that MDA had formed. However, addition of larger concentrations of Fe(III)2 (SO4 )3 to NaTBA decreased ¯uorescence. This quenching of ¯uorescence would provide a clue that the increase in absorbance at 532 nm following the addition of Fe(III) is not due to the formation of MDA. To obtain additional information concerning the stoichiometry of the reaction of Cu(II) and Fe(III) with NaTBA, solutions of 3.5 mM Cu(II) or 2 mM Fe(III) were allowed to react with 0±10 mM NaTBA. A linear increase in absorbance was observed as shown in Fig. 4, for the addition of 0±7 mM NaTBA to the 3.5 mM Cu(II) solution with a dramatic increase in absorbance when the 2 : 1 ratio of TBA: Cu(II) was
Fig. 4. Absorbance for solutions of 3.5 mM Cu(II)Cl2 to which was added increasing concentrations of NaTBA at room temperature (d), after heating for 5 min at 100°C (s), and after centrifuging the heated solution (D).
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L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
Fig. 5. Absorbance for solutions of 2 mM Fe(III)Cl3 to which was added increasing concentrations of NaTBA at room temperature (d), after heating for 5 min at 100°C (s) and after centrifuging the heated solution (D).
exceeded suggesting the formation of a new species which also absorbed at 532 nm. Centrifuging these solutions revealed the formation of a precipitate, and the loss of the chromophore observed following the addition of NaTBA to Cu(II)Cl2 at room temperature (Fig. 4). Heating these solutions for 5 min at 100°C caused a marked increase in absorbance, and precipitate formation again prevented the measurement of absorbance when the concentration of NaTBA exceeded 3 mM (Fig. 4). A linear increase in absorbance was also observed for the addition of 2 mM Fe(III) throughout the entire concentration range of 0±10 mM NaTBA, as shown in Fig. 5, without precipitate formation. Heating these solutions for 5 min at 100°C further increased their absorbance for the addition 0±10 mM NaTBA with precipitate formation as the ratio of NaTBA: Fe(III) exceeded 3 : 1 (Fig. 5). With the addition of more than three equivalents of NaTBA further precipitation occurred with the loss of the chromophore accounting for the 532 nm absorbance observed for the addition of Fe(III) to NaTBA at room temperature (Fig. 5). These precipitates are either complexes of TBA shown in Scheme 3 or TBA disul®de shown in Scheme 4.
4. Discussion There have been many suggestions that Cu and Fe cause lipid and other tissue component oxidations via Fenton-like or Fenton chemistry respectively based upon results obtained using the TBA assay. This assay is based on the reaction of TBA with MDA, which is a product of unsaturated lipid and other tissue component
oxidations. The resulting conjugated aromatic chromophore of the bis-TBA-MDA adduct has a distinct absorbance at 532 nm and ¯uorescence at 553 nm. Copper or Fe complexes of TBA and/or a Cu or Fe complex of TBA disul®de, the oxidation product of TBA, possess a chromophore absorbing at 532 nm. Heating these mixtures of Cu or Fe and TBA causes a marked increase in absorbance at 532 nm. Butanol extracts of Cu- or FeTBA mixtures also absorbed at 532 nm and it is of interest to note that this absorbance increased with time for Cu-TBA complexes while there was a decrease in absorbance for Fe-TBA complexes in butanol. Consequently, assertions that Cu and Fe cause lipid and other tissue component oxidations based upon 532 nm absorbance in the TBA assay are false. Failure to control for the 532 nm absorbance as being due to the addition of Cu or Fe to the TBA assay system will lead to the false conclusion that the bis-TBA-MDA adduct has formed as a result of lipid and other tissue component oxidations. The use of the TBA assay to detect MDA as described by Jamero [23], who has pointed out other interferences, would enable TBA-test practioners to accurately determine this lipid oxidation product even though the yield of MDA is very low relative to the amount of fatty acid peroxide formed. Interferences due to the presence of physiological amounts of ionically bonded Fe and/or Cu, 10ÿ23 or 10ÿ18 M, respectively [67], in plasma or still smaller amounts in solid tissues are not likely and can be avoided with the use of appropriate control samples. It is also of interest to note that while excited singlet states of Cu-TBA complex(es) do undergo a ¯uorescent emission at 553 nm, the Fe-TBA complex(es) do not undergo a ¯uorescent emission at 553 nm and additions of Fe(III) quench the weak ¯uorescence due to TBA. Since many researchers only measure absorbance and fail to recognize this absorbance as being due to complex formation they miss this lack of ¯uorescence clue that the measured 532 nm absorbance is not due to the bis-TBA-MDA adduct. Studies of Fe and Cu metabolism can be very bene®cial in understanding normal and disease states. Past research eorts attempted to show that Cu or Fe react with inappropriately large concentrations of hydrogen peroxide to produce unsubstantiated HOá. Hydroxyl radical as well as singlet oxygen (1 O2 ), hydroperoxyl radical (HOO), as well as superoxide (Oÿ 2 á) can cause lipid and other tissue component oxidations. It is widely accepted that these substantiated free radicals cause disease pathology and may have a major role in the underlying disease process of many disease states. However, addition of inorganic forms of Cu or Fe to biochemical systems intended to demonstrate oxidation of tissue components do not necessarily cause the formation of HO´ to account for the observed increase in 532 nm absorbance or 553 nm ¯uorescence as has been suggested by many researchers based upon TBA assay results. In addition, both Cu and Fe are extremely ecient scavengers of
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
HO´ ; HOO; Oÿ 2 , with rates of removal of the order of 108 ±109 moleÿ1 sÿ1 for Cu(II) salts and Cu(II) complexes and 109 ±1010 moleÿ1 sÿ1 for Fe(III) salts and Fe(III) complexes ([70,72] and references there in) and the formation of HO´ by ionically bonded Cu or Fe in the presence of H2 O2 has been seriously questioned [60]. Interestingly, the disappearance of ¯uorescence at 553 nm following the addition of Fe to NaTBA solutions would reveal the absence of the bisTBA-MDA adduct when using the TBA assay in the presence of Fe. The formation of H2 O2 , 1 O2 , HOOá, and HOá are associated with the accumulation of Oÿ 2 . Accumulation of Oÿ 2 is associated with decreases in both cytosolic and extracellular Cu2 Zn2 -superoxide dismutases and hydrogen peroxide accumulates with less than normal concentrations of catalase, an Fe-dependent enzyme. The accumulation of H2 O2 in the presence of Oÿ 2 also leads to the formation of 1 O2 and HO´ . Since de®ciencies in Cu2 Zn2 SODs and catalase occur with Cu and Fe de®ciencies it is most likely that Cu and Fe de®ciencies have a role in the underlying oxygen radical-mediated disease processes and not the presence of ionically bonded Cu and/or Fe, which are not measurable in any tissue. Throughout the recent past there have been many reports [1±65] that Cu and/or Fe cause ``HOá-mediated oxidations'' based upon TBA assay results. These now appear to be ¯awed due to a failure to control for the formation of Cu- and/or Fe-TBA complexes and/or oxidation of TBA to its disul®de by Cu or Fe and subsequent complex formation. Had these control studies been done with measurement of both absorbance at 532 nm and ¯uorescence at 553 nm, as originally suggested by Yagi [71], for authentic bis-TBA-MDA adduct, interferences we are reporting would have been noted. The published literature does contain clues relating to Cu and Fe interferences in the TBA assay [3,5,24,73,74] including Cu-TBA [73] and Fe-TBA [3] complex formation and what may have been oxidation products of TBA. Some literature reporting ``HOá-mediated oxidation'' in inappropriately Cu(II) and/or Fe(III) perturbed systems based upon the TBA assay data obtained by themselves or others also report bene®cial eects of Cudependent proteins such as antioxidant activity of Ceruloplasmin and other Cu complexes [15,19,26,28,29, 55,65,75], inhibition of Oÿ 2 á mediated Fenton chemistry by cytosolic Cu2 Zn2 SOD [54,76], or extracellular Cu2 , Zn2 , SOD [77], prevention of lipoprotein oxidation with adequate dietary Cu intake [27], Cu-mediated prevention of in¯ammation- and radiation-induced lipid peroxidation [75±78], prevention of oxidative damage to skeletal muscle in exercise with derivatized Cu2 Zn2 SOD [79], and scavenging of HOá by copper complexes [80]. These bene®cial eects are consistent with the antiin¯ammatory, antiulcer, anticonvulsant, anticancer, anticarcinogenic, antimutagenic, antidiabetic, radioprotectant and radiorecovery activities of Cu complexes as well as prevention of ischemia-reperfusion injury with
223
Cu complexes ([72] and references therein). The radioprotectant activity of an Fe complex is evidence that small molecular mass Fe complexes also bene®cially aect the outcome of this massive in¯ammatory disease known to be caused as a result of radiation-induced HO´ and Oÿ 2 formation [81]. A serious question is raised with regard to the modeled disease states [1±65] including the specious suggestion that Wilson's disease is ``copper toxicosis'' [82] wherein the suggested pathology is due to Cu or Fe mediated HOá formation. Since ionically bonded Cu and Fe do not exist in any tissue in quantities measurable with existing equipment and the use of very large concentrations of other agents such as H2 O2 which would be lethal to cells, the relevance of these studies must be examined. The observed increase in what is interpreted as oxygen radical-mediated oxidative distruction using the TBA assay of a system to which ionically bonded Cu or Fe compounds are added may be due to the indiscriminate bonding of Cu or Fe to components of the system causing chemical and physical changes of these components that account for these false interpretations. Since small molecular mass Cu and Fe complexes have SOD-mimetic and catalase-mimetic activities ([69,72] and references therein) and Cu complexes down-regulate nitric oxide synthase [83] it seems reasonable to suggest that these complexes may be useful in preventing these disease states. The etiology of atherosclerosis and other in¯ammatory diseases listed in the Introduction Section of this manuscript may indeed result from the formation of HOá, HOOá, and 1 O2 due to the accumulation of Oÿ 2 in relevant tissues. This accumulation of Oÿ 2 resulting from Cu de®ciency and the consequent decrease in the Cu-dependent SOD or the accumulation of H2 O2 resulting from Fe de®ciency and the consequent decrease in Fe-dependent catalase oer insights into the mechanism of disease onset and development. This then begs the question as to why small molecular mass Cu and Fe complexes, which are bioavailable forms of Cu and Fe, should be used to provide Cu and Fe for de novo synthesis of Cu2 Zn2 -SOD or catalase as well as other Cu- or Fe-dependent enzymes. Finally, with the inappropriate addition of ionically bonded Cu(II) and/or Fe(III) to biological systems there is a failure to recognize Cu(II), and/or Fe(III), catalyzed carboxylic and phosphate ester hydrolyses as well as amide hydrolysis [84,85], which are well known and do not involve HOá formation.
Acknowledgements We are indebted to Chancellor Harry P. Ward for a Special Research Grant and the Essential Metalloelement Analysis Laboratory for funding and to James Masuoka for his critical comments of our manuscript.
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Formation of Cu or Fe thiobarbiturate complexes interfere with the determination of malondialdehyde Lily Zhou, John R.J. Sorenson
*
Division of Medicinal Chemistry, College of Pharmacy, University of Arkansas for Medical Sciences Campus, Little Rock, Arkansas 72205, USA Received 16 July 1998; received in revised form 6 October 1998; accepted 22 October 1998
Abstract This research was conducted to determine whether or not Cu(II) and Fe(III) form TBA complexes that absorb and ¯uoresce at the same wavelengths as the bis-TBA-MDA adduct and support a false conclusion that Cu(II) and Fe(III) cause lipid and other tissue component oxidations as evidenced by the apparent formation of the bis-TBA-MBA adduct. Additions of Cu(II) or Fe(III) to Na TBA gave concentration related increases in absorbance at 532 nm, the wavelength of maximum absorbance of the bis-TBAMDA adduct. These absorbance spectra demonstrate that the addition of aqueous solutions of Na TBA to systems containing added Cu(II) and/or Fe(III) and their butanol extracts will support the false conclusion that Cu(II) and/or Fe(III) caused lipid and other tissue component oxidations. Fluorescence at 553 nm, the maximum for ¯uorescence of the bis-TBA-MDA adduct, increased as the concentration of Cu(II) increased but decreased with the addition of Fe(III). Fluorescence obtained following the addition of Cu(II) to Na TBA will support the false conclusion that Cu(II) causes lipid and other tissue component oxidations while the lack of ¯uorescence following the addition of Fe(III) will not support this false conclusion. These studies show that the presence of Cu(II) or Fe(III) in TBA assay systems lead to the specious interpretation that oxidations occurred due to the formation of HO´ radical via Cu(II)- or Fe(III)-mediated Fenton chemistry. Ó 1998 Elsevier Science Inc. All rights reserved. Keywords: Copper; Iron; Thiobarbituric acid; Absorbance; Fluorescence; Malondialdehyde
1. Introduction Unsaturated lipid [1±40], lipoprotein [27,30,35,37, 38,41±50], deoxyribose or DNA [15±18,24,25,31,35,50± 60], protein [18,25,27,30,31,61±63], carbohydrate [51,56], bile pigment [53], lens crystallins [61,64], catecholamines [34], and mylein [30] oxidative degradations are suggested to have a major pathological role in many disease states including arthritidies [15±17,19,53,56,62], atherosclerosis [27,33,35,37,40,41,43,45,47±49], central neuronal cell death [31,39], hemolysis [8,28,65], thrombosis [43], myocardial infarction [41,43,44], aceruloplasminemia [39], renal insuciency [32], diabetes [32,33], cardiovascular disease [27,37,43,44], peripheral vascular disease [37], pancreatitis [32], liver cirrhosis [32,63], cancer [32], angioplasty injury [48], radiation injury [7,8,18,50,56], ischemia-reperfusion injury [31,33,64], as well as Parkinson's [30,31,34], Alzheimer's [30,31], Menkes' [63], and Wilson's disease [15,17,33,39]. *
Corresponding Author. E-mail: [email protected]
These degradations and disease states are often modeled as in vitro biological systems by addition of hydrogen peroxide (H2 O2 ), vitamin C, or thiols and Fe and/or Cu and sometimes heating up to 100°C. These conditions are hypothesized to lead to the formation of an aggressive oxidant most frequently suggested to be hydroxyl radical (HO´ ) formed via Fenton chemistry [66]. Lower oxidation states of Cu(I) or Fe(II) in tissues are suggested to cause homolytic cleavage of hydrogen peroxide to yield HO´ ; Fe(II) or Cu(I) + H2 O2 ® Fe(III) or Cu (II) + HO´ OHÿ . The assumed formation of HO´ is then suggested to cause the formation of malondialdehyde (MDA), as shown in Scheme 1, which is then assumed to react with thiobarbituric acid (TBA) to account for the increase in measured absorbance at 532 nm and/or ¯uorescence at 553 nm. Perusal of the literature [1±65] revealed that nearly all experimental paradigms intended to produce HO´ mediated destruction involved the addition of something of the order of 10ÿ6 to 10ÿ3 M ionically bonded Cu(II) or Fe(III) to modeled biological systems. The
0162-0134/98/$ ± see front matter Ó 1998 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 1 0 0 8 3 - 1
218
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
Scheme 1.
highest concentration found was 0.3 M [48]. Since calculated concentrations of ionically bonded Cu and Fe in plasma are of the order of 10ÿ18 and 10ÿ23 M [67], respectively, and are too small to be measured with any exiting analytical method, additions of much larger amounts of ionically bonded Cu or Fe seem to lack relevance. Amounts of ionically bonded Cu and Fe in solid tissues are likely to be still lower due to a greater number of bonding sites. The remaining Cu and Fe found in all tissues exhists as stable complexes including Cu- and Fe-dependent enzymes, and these coordinate-covalently bonded forms of Cu and Fe do not represent ionically-bonded redox-active forms of these metalloelements that are likely to be serious threats to cells as HO generators. As a result, it was questioned as to whether or not additions of ionically bonded forms of Cu and Fe led to indiscriminate bonding of these metalloelements to components of these biological systems and had a role in causing the reported system de®cits that were interpreted as oxidative degradations. Another curious observation was the use of concentrations of H2 O2 ranging up to 33.3 M when the estimated concentration of H2 O2 in normal cells is of the order of 10ÿ8 M [68]. It was questioned as to whether or not these high concentrations were relevant since it is unlikely that cells containing much more than 10ÿ8 M H2 O2 would remain viable. The need for the use of large concentrations of H2 O2 in these experiments may arise from the fact that both Cu(II) and Fe(III) have catalasemimetic activity [69], and can convert lower concentrations of hydrogen peroxide to triplet state oxygen and water at rates of the order of 109 molÿ1 sÿ1 , and Cu(I) and Cu(II) as well as Fe(II) and Fe(III) are excellent HO´ scavengers with rates of reduction of hydroxyl radical, HO´ ® HOÿ , of the order of 1010 molÿ1 sÿ1 [70] with oxidation of these metalloelements to the next
higher oxidation state: Cu(II) and Cu(III) or Fe(III) and Fe(IV). Authentic HO´ generation is known to lead to the formation of MDA [2,23]. The TBA assay has been widely used as a test for MDA produced by lipid and other tissue component oxidations in modeled biological systems. This assay is based on the reaction of TBA with the bifunctional MDA to yield a bis-TBA-MDA adduct [2,23]. The resulting conjugated aromatic chromophore shown in Scheme 2 has a characteristic absorbance maximum at 532 nm and ¯uorescence maximum at 553 nm [71]. In the light of the above, it occurred to us that additions of Cu and Fe to modeled biological systems may also produce an absorbance at 532 nm and an emission at 553 nm following the addition of TBA due to the formation of Cu or Fe complexes of TBA, which may be falsely interpreted as tissue component oxidation by Cu or Fe based upon the TBA assay. Unfortunately these TBA assay results reported by many are not controlled for the addition of Cu or Fe as they may eect the TBA assay by increasing the observed absorbance due to formation of Cu or Fe complexes of TBA: Cu(II)(thiobarbiturate)2 or Fe(III)-(thiobarbiturate)3 , as shown in Scheme 3, and/or the oxidation of TBA to its disul®de by Cu(II) or Fe(III) and the formation of Cu(II) or Fe(III) complexes of the disul®de, as shown below in Scheme 4, where n is the number of TBA ligands and nI is the oxidation state of Cu(I or II) or Fe(II or III). To examine the possibility that ionically bonded Cu and Fe compounds form these complexes and/or TBA disul®de and cause a positive interference with the determination of the bis-MDA-TBA adduct, a product of lipid and other tissue component oxidations produced by HO,both absorbance and ¯uorescence spectra were obtained for mixtures of ionically bonded Cu and Fe compounds and TBA. 2. Materials and methods Cupric Chloride, [Cu(II)C12 ] (Spectrum Chemical Corp, A. C. S. Reagent), Ferric Chloride, [Fe(III)C13 ] (Mallinckrodt Chemical Works, Analytical Reagent), Ferric Sulfate, [Fe(III)2 (SO4 )3 ] (Alfa Products, Reagent), Sodium Hydroxide, (NaOH) (Aldrich Chemical Company, A. C. S. Reagent), Thiobarbituric Acid,
Scheme 2.
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
219
Scheme 3.
Scheme 4.
(TBA) (Sigma Chemical Co, 98% pure) were used without further puri®cation. Butanol or deionized water were used to prepare solutions. An IEC HN-S II centrifuge was used to centrifuge turbid solutions. One centimeter quartz cuvettes were used for spectrophotometric and ¯uorescence measurements. A HewlettPackard 8452a Diode Array Spectrophotometer with a Vectna E5 Computer and Printer was used to measure and record absorbance. A Perkin Elmer Luminescence Spectrometer was used to measure ¯uorescence from 200 to 800 nm. All glassware (beakers, Erlenmeyer ¯asks, volumetric ¯asks, graduated cylinders, medium porosity sintered fritted glass ®lter funnels, and suction ¯asks) were thoroughly cleaned with Citronox (Alconox Inc.), Acetone, or 10% Hydrochloric Acid (HCl). Other equipment including: spatulas, test tubes, Pasteur pipettes, and disposable tips for hand held pipettes were metal free. To prepare 100 ml of a 50 mM NaOH solution, 0.2 gm (5 mmol) of NaOH was dissolved in 100 ml of deionized H2 O. To prepare 100 ml of a 14.3 mM NaOH solution, 0.057 gm (1.43 mmol) of NaOH was dissolved in 100 ml of deionized water. To prepare 100 ml of a 70 mM TBA solution, 1.008 gm (7 mmol) of TBA was dissolved in 100 ml of 50 mM NaOH solution and ®ltered through a sintered glass funnel. To prepare 100 ml of a 7 mM solution of Cu(II)Cl2 (H2 O)2 , 0.229 gm (0.7 mmol) was dissolved in 100 ml of deionized water. To prepare 100 ml of a 35 mM solution of cupric chloride, 0.597 gm (3.5 mmol) of Cu(II)C12 (H2 O)2 was dissolved in 100 ml of deionized H2 O. To prepare 100 ml of a 23 mM solution of Fe(III)C13 , 0.622 gm (2.3 mmol) of Fe(III)C13 (H2 O)6 was dissolved in 100 ml of deionized H2 O. To prepare 100 ml of 4 mM solution of Fe(III)Cl3 , 0.1081 gm (0.4 mmol of Fe(III)Cl2 (H2 O)6 was dissolved in 100 ml of deionized water. To prepare
100 ml of a 23 mM solution of Fe(III)2 (SO4 )3 , 1.044 gm (2.3 mmol) of Fe(III)2 (SO4 )3 (H2 O)3 was dissolved in 100 ml of deionized H2 O and ®ltered through a sintered glass funnel. To obtain spectra for mixtures of Cu(II)C12 or Fe(III)2 (SO4 )3 [or Fe(III)C13 ] and TBA, absorbance and ¯uorescence were set at zero with a solution of 0.15 ml of 50 mM NaOH in 2.5 ml of deionized water. Fluorescence measurements were done in triplicate. The amount of TBA in these solutions was held constant and the amount of Cu(II)C12 or Fe(III)Cl 3 or Fe(III)2 (SO4 )3 added was varied as shown in Tables 1 and 2 respectively. A volume of deionized H2 O was added to the cuvette prior to the addition of TBA and either Cu(II)C12 or Fe(III)Cl3 or Fe(III)2 (SO4 )3 with thorough mixing to maintain a constant volume in the cuvette. The following procedures were used to obtain spectra of butanol extracted chromophore produced with the mixture of Cu(II) or Fe(III) and NaTBA solutions. 2 ml of 70 mM TBA in 50 mM NaOH and 2 ml of 35 mM Cu(II)C12 were pipetted into a metal-free polypropylene culture tube. Five ml of butanol was then added and the Table 1 Volumes and ®nal concentrations of TBA and Cu(II)Cl2 used to prepare solutions for absorbance and ¯uorescence measurements
1. 2. 3. 4. 5.
TBA (®nal concentration)
Cu(II)C12 (®nal concentration)
Deionized water
0.30 0.30 0.30 0.30 0.30
0.00 0.15 0.20 0.25 0.30
2.70 2.55 2.50 2.45 2.40
ml ml ml ml ml
(7 (7 (7 (7 (7
mM) mM) mM) mM) mM)
ml ml ml ml ml
(0.00 (1.75 (2.33 (2.92 (3.50
mM) mM) mM) mM) mM)
ml ml ml ml ml
220
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
Table 2 Volumes and ®nal concentrations of TBA and Fe(III)Cl2 or Fe(III)2 (SO4 )3 used to prepare solutions for absorbance and ¯uorescence measurements
1. 2. 3. 4. 5.
TBA (®nal concentration)
Fe(III)Cl3 or Fe(III)2 (SO4 )3 (®nal concentration)
Deionized H2 O
1.50 1.50 1.50 1.50 1.50
0.03 ml (0.23 mM) 0.06 ml (0.46 mM) 0.125 ml (0.96 mM) 0.25ml (1.92 mM) 0.50 ml (3.83 mM)
1.47 ml 1.44 ml 1.375 ml 1.25 ml 1.00 ml
ml ml ml ml ml
(35 (35 (35 (35 (35
mM) mM) mM) mM) mM)
mixture stirred with a vortex mixer for 5 minutes. The same procedure was repeated using 2 ml of 70 mM TBA in 50 mM NaOH, 2 ml of 23 mM Fe(III)C13 , and 5 ml of butanol. The butanol layer was removed from each mixture using metal-free disposable Pasteur pipettes and placed in two separate polypropylene culture tubes. Ultraviolet±Visible spectra were obtained for each butanol solution after setting the absorbance at zero using 3 ml of butanol. To examine stoichiometries of the reaction of Cu(II)Cl2 and Fe(III)Cl3 with TBA, concentrations of Cu(II)Cl2 and Fe(III)Cl3 were held constant and the concentration of NaTBA added was varied as shown in Tables 3 and 4, respectively. All spectral measurements were performed in triplicate. To examine the in¯uence of heating on the appearance of the chromophore, solutions represented in Tables 3 and 4 were heated at 100°C for 5 min and spectra recorded. This heating procedure caused precipitate formation in some solutions. When a precipitate formed the solution was centrifuged for 10 min at about 2000 revolutions per minute and a spectrum obtained for the supernatant. All determinations for the mixture of Cu(II)Cl2 or Fe(III)Cl3 with NaTBA were performed on the same day to avoid time-dependent variations. All measurements were done in triplicate.
Table 4 Volumes and concentrations of Fe(III)Cl3 and TBA solutions used to examine the stiochiometry of chromophore formation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
TBA (®nal concentration)
Fe(III)Cl3 (®nal concentration)
Deionized water
1.50 1.35 1.20 1.05 0.90 0.75 0.60 0.45 0.30 0.15 0.00
1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50
0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50
ml ml ml ml ml ml ml ml ml ml ml
(10 mM) (9 mM) (8 mM) (7 mM) (6 mM) (5 mM) (4 mM) (3 mM) (2 mM) (1 mM) (0 mM)
ml ml ml ml ml ml ml ml ml ml ml
(2 (2 (2 (2 (2 (2 (2 (2 (2 (2 (2
mM) mM) mM) mM) mM) mM) mM) mM) mM) mM) mM)
ml ml ml ml ml ml ml ml ml ml ml
3. Results Fig. 1 shows the increase in absorbance at 532 nm, which would be falsely interpreted as increased MDA formation, when increasing concentrations of Cu(II)C12 ranging from 0.00 to 3.50 mM were added to a 7 mM solution of NaTBA. There was a concentration related increase in absorbance and intensity of greenish-yellow color when concentrations of 2.3 mM Cu(II)Cl2 and larger were added to 7 mM NaTBA. An increase in the concentration of Cu(II)Cl2 above 3.5 mM resulted in the formation of a greenish-yellow precipitate, suggesting the formation of a mixture of a Cu(I) thiol and Cu(II) oxygen-nitrogen chelates of TBA or TBA disul®de. The increase in absorbance at 532 nm and intensity of red-wine color, consistent with the false interpretation that MDA formed, when increasing concentrations of
Table 3 Volumes and concentrations of Cu(II)Cl2 and TBA solutions used to examine the stoichiometry of chromophore formation
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
TBA (®nal concentration)
Cu(II)Cl2 (®nal concentration)
Deionized water
1.50 1.35 1.20 1.05 0.90 0.75 0.60 0.45 0.30 0.15 0.00
1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50
0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50
ml ml ml ml ml ml ml ml ml ml ml
(10 mM) (9 mM) (8 mM) (7 mM) (6 mM) (5 mM) (4 mM) (3 mM) (2 mM) (1 mM) (0 mM)
ml ml ml ml ml ml ml ml ml ml ml
(3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5 (3.5
mM) mM) mM) mM) mM) mM) mM) mM) mM) mM) mM)
ml ml ml ml ml ml ml ml ml ml ml
Fig. 1. Increase in absorbance at 532 nm for addition of Fe(III)Cl3 to 35 mN NaTBA or Cu(II)Cl2 to 7 mM NaTBA.
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
221
Fig. 3. Increase in ¯ourescence at 553 nm for addition of Cu(II)Cl2 to 7mM NaTBA or decrease in ¯oursence at 553 nm for addition of Fe(III)2 (SO4 )3 to 35 mM NaTBA.
Fig. 2. Time dependent incease in abosorbance at 532 nm for a butanol extract of a 5.11 mM Fe(III)Cl3 -15.56 mM NaTBA solution or a 7.78 mM Cu(II)Cl2 -15.56 mM NaTBA solution.
Fe(III)Cl3 ranging from 0.00 to 3.83 mM were added to a 35 mM solution of NaTBA is shown in Fig. 1. No precipitate formed when these concentrations of Fe(III)Cl3 were added to 35 mM NaTBA, which was ®ve times as concentrated as the NaTBA solution used for additions of Cu(II)Cl2 . Ultraviolet±Visible spectrophotometric measurements were also made for a butanol extract of the mixture of 7.78 mM Cu(II)Cl2 and 15.56 mM NaTBA and the mixture of 5.11 mM Fe(III)Cl3 and 15.56 mM NaTBA. Following extraction of these mixtures it was noticed that absorbances of these extracts changed over a period of 6 h. The absorbance at 532 nm increased as shown in Fig. 2 for the butanol extract of the Cu(II)Cl2 ± NaTBA solution through the period of this experiment while the 532 nm absorbance for the butanol extract of the Fe(III)Cl3 ±NaTBA solution decreased over the ®rst hour it did not change signi®cantly over the next 5 h (Fig. 2). The observed increase in absorbance for both butanol extracts would also be falsely interpreted as being due to the formation of MDA. The linear increase in ¯uorescence at 553 nm observed when increasing concentrations of Cu(II)Cl2 ranging from 0.00 to 3.50 mM were added to a 7 mM solution of NaTBA is shown in Fig. 3. This increase in ¯uorescence, would also lead to the false interpretation that MDA had been formed. Fig. 3 shows the ¯uorescence observed at 553 nm when increasing concentrations of Fe(III)2 (SO4 )3 ranging from 0.00 to 3.83 mM were added to a 35 mM solution of NaTBA. The ¯uorescence observed following the addition of 0.23 mM Fe(III)2 (SO4 )3 , 0.46 mM Fe(III), which was less than the ¯uorescence observed for the 35 mM solution of NaT-
BA, 0.07 ergs sÿ1 , still is consistent with the false notion that MDA had formed. However, addition of larger concentrations of Fe(III)2 (SO4 )3 to NaTBA decreased ¯uorescence. This quenching of ¯uorescence would provide a clue that the increase in absorbance at 532 nm following the addition of Fe(III) is not due to the formation of MDA. To obtain additional information concerning the stoichiometry of the reaction of Cu(II) and Fe(III) with NaTBA, solutions of 3.5 mM Cu(II) or 2 mM Fe(III) were allowed to react with 0±10 mM NaTBA. A linear increase in absorbance was observed as shown in Fig. 4, for the addition of 0±7 mM NaTBA to the 3.5 mM Cu(II) solution with a dramatic increase in absorbance when the 2 : 1 ratio of TBA: Cu(II) was
Fig. 4. Absorbance for solutions of 3.5 mM Cu(II)Cl2 to which was added increasing concentrations of NaTBA at room temperature (d), after heating for 5 min at 100°C (s), and after centrifuging the heated solution (D).
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Fig. 5. Absorbance for solutions of 2 mM Fe(III)Cl3 to which was added increasing concentrations of NaTBA at room temperature (d), after heating for 5 min at 100°C (s) and after centrifuging the heated solution (D).
exceeded suggesting the formation of a new species which also absorbed at 532 nm. Centrifuging these solutions revealed the formation of a precipitate, and the loss of the chromophore observed following the addition of NaTBA to Cu(II)Cl2 at room temperature (Fig. 4). Heating these solutions for 5 min at 100°C caused a marked increase in absorbance, and precipitate formation again prevented the measurement of absorbance when the concentration of NaTBA exceeded 3 mM (Fig. 4). A linear increase in absorbance was also observed for the addition of 2 mM Fe(III) throughout the entire concentration range of 0±10 mM NaTBA, as shown in Fig. 5, without precipitate formation. Heating these solutions for 5 min at 100°C further increased their absorbance for the addition 0±10 mM NaTBA with precipitate formation as the ratio of NaTBA: Fe(III) exceeded 3 : 1 (Fig. 5). With the addition of more than three equivalents of NaTBA further precipitation occurred with the loss of the chromophore accounting for the 532 nm absorbance observed for the addition of Fe(III) to NaTBA at room temperature (Fig. 5). These precipitates are either complexes of TBA shown in Scheme 3 or TBA disul®de shown in Scheme 4.
4. Discussion There have been many suggestions that Cu and Fe cause lipid and other tissue component oxidations via Fenton-like or Fenton chemistry respectively based upon results obtained using the TBA assay. This assay is based on the reaction of TBA with MDA, which is a product of unsaturated lipid and other tissue component
oxidations. The resulting conjugated aromatic chromophore of the bis-TBA-MDA adduct has a distinct absorbance at 532 nm and ¯uorescence at 553 nm. Copper or Fe complexes of TBA and/or a Cu or Fe complex of TBA disul®de, the oxidation product of TBA, possess a chromophore absorbing at 532 nm. Heating these mixtures of Cu or Fe and TBA causes a marked increase in absorbance at 532 nm. Butanol extracts of Cu- or FeTBA mixtures also absorbed at 532 nm and it is of interest to note that this absorbance increased with time for Cu-TBA complexes while there was a decrease in absorbance for Fe-TBA complexes in butanol. Consequently, assertions that Cu and Fe cause lipid and other tissue component oxidations based upon 532 nm absorbance in the TBA assay are false. Failure to control for the 532 nm absorbance as being due to the addition of Cu or Fe to the TBA assay system will lead to the false conclusion that the bis-TBA-MDA adduct has formed as a result of lipid and other tissue component oxidations. The use of the TBA assay to detect MDA as described by Jamero [23], who has pointed out other interferences, would enable TBA-test practioners to accurately determine this lipid oxidation product even though the yield of MDA is very low relative to the amount of fatty acid peroxide formed. Interferences due to the presence of physiological amounts of ionically bonded Fe and/or Cu, 10ÿ23 or 10ÿ18 M, respectively [67], in plasma or still smaller amounts in solid tissues are not likely and can be avoided with the use of appropriate control samples. It is also of interest to note that while excited singlet states of Cu-TBA complex(es) do undergo a ¯uorescent emission at 553 nm, the Fe-TBA complex(es) do not undergo a ¯uorescent emission at 553 nm and additions of Fe(III) quench the weak ¯uorescence due to TBA. Since many researchers only measure absorbance and fail to recognize this absorbance as being due to complex formation they miss this lack of ¯uorescence clue that the measured 532 nm absorbance is not due to the bis-TBA-MDA adduct. Studies of Fe and Cu metabolism can be very bene®cial in understanding normal and disease states. Past research eorts attempted to show that Cu or Fe react with inappropriately large concentrations of hydrogen peroxide to produce unsubstantiated HOá. Hydroxyl radical as well as singlet oxygen (1 O2 ), hydroperoxyl radical (HOO), as well as superoxide (Oÿ 2 á) can cause lipid and other tissue component oxidations. It is widely accepted that these substantiated free radicals cause disease pathology and may have a major role in the underlying disease process of many disease states. However, addition of inorganic forms of Cu or Fe to biochemical systems intended to demonstrate oxidation of tissue components do not necessarily cause the formation of HO´ to account for the observed increase in 532 nm absorbance or 553 nm ¯uorescence as has been suggested by many researchers based upon TBA assay results. In addition, both Cu and Fe are extremely ecient scavengers of
L. Zhou, J.R.J. Sorenson / J. Inorg. Biochem. 72 (1998) 217±225
HO´ ; HOO; Oÿ 2 , with rates of removal of the order of 108 ±109 moleÿ1 sÿ1 for Cu(II) salts and Cu(II) complexes and 109 ±1010 moleÿ1 sÿ1 for Fe(III) salts and Fe(III) complexes ([70,72] and references there in) and the formation of HO´ by ionically bonded Cu or Fe in the presence of H2 O2 has been seriously questioned [60]. Interestingly, the disappearance of ¯uorescence at 553 nm following the addition of Fe to NaTBA solutions would reveal the absence of the bisTBA-MDA adduct when using the TBA assay in the presence of Fe. The formation of H2 O2 , 1 O2 , HOOá, and HOá are associated with the accumulation of Oÿ 2 . Accumulation of Oÿ 2 is associated with decreases in both cytosolic and extracellular Cu2 Zn2 -superoxide dismutases and hydrogen peroxide accumulates with less than normal concentrations of catalase, an Fe-dependent enzyme. The accumulation of H2 O2 in the presence of Oÿ 2 also leads to the formation of 1 O2 and HO´ . Since de®ciencies in Cu2 Zn2 SODs and catalase occur with Cu and Fe de®ciencies it is most likely that Cu and Fe de®ciencies have a role in the underlying oxygen radical-mediated disease processes and not the presence of ionically bonded Cu and/or Fe, which are not measurable in any tissue. Throughout the recent past there have been many reports [1±65] that Cu and/or Fe cause ``HOá-mediated oxidations'' based upon TBA assay results. These now appear to be ¯awed due to a failure to control for the formation of Cu- and/or Fe-TBA complexes and/or oxidation of TBA to its disul®de by Cu or Fe and subsequent complex formation. Had these control studies been done with measurement of both absorbance at 532 nm and ¯uorescence at 553 nm, as originally suggested by Yagi [71], for authentic bis-TBA-MDA adduct, interferences we are reporting would have been noted. The published literature does contain clues relating to Cu and Fe interferences in the TBA assay [3,5,24,73,74] including Cu-TBA [73] and Fe-TBA [3] complex formation and what may have been oxidation products of TBA. Some literature reporting ``HOá-mediated oxidation'' in inappropriately Cu(II) and/or Fe(III) perturbed systems based upon the TBA assay data obtained by themselves or others also report bene®cial eects of Cudependent proteins such as antioxidant activity of Ceruloplasmin and other Cu complexes [15,19,26,28,29, 55,65,75], inhibition of Oÿ 2 á mediated Fenton chemistry by cytosolic Cu2 Zn2 SOD [54,76], or extracellular Cu2 , Zn2 , SOD [77], prevention of lipoprotein oxidation with adequate dietary Cu intake [27], Cu-mediated prevention of in¯ammation- and radiation-induced lipid peroxidation [75±78], prevention of oxidative damage to skeletal muscle in exercise with derivatized Cu2 Zn2 SOD [79], and scavenging of HOá by copper complexes [80]. These bene®cial eects are consistent with the antiin¯ammatory, antiulcer, anticonvulsant, anticancer, anticarcinogenic, antimutagenic, antidiabetic, radioprotectant and radiorecovery activities of Cu complexes as well as prevention of ischemia-reperfusion injury with
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Cu complexes ([72] and references therein). The radioprotectant activity of an Fe complex is evidence that small molecular mass Fe complexes also bene®cially aect the outcome of this massive in¯ammatory disease known to be caused as a result of radiation-induced HO´ and Oÿ 2 formation [81]. A serious question is raised with regard to the modeled disease states [1±65] including the specious suggestion that Wilson's disease is ``copper toxicosis'' [82] wherein the suggested pathology is due to Cu or Fe mediated HOá formation. Since ionically bonded Cu and Fe do not exist in any tissue in quantities measurable with existing equipment and the use of very large concentrations of other agents such as H2 O2 which would be lethal to cells, the relevance of these studies must be examined. The observed increase in what is interpreted as oxygen radical-mediated oxidative distruction using the TBA assay of a system to which ionically bonded Cu or Fe compounds are added may be due to the indiscriminate bonding of Cu or Fe to components of the system causing chemical and physical changes of these components that account for these false interpretations. Since small molecular mass Cu and Fe complexes have SOD-mimetic and catalase-mimetic activities ([69,72] and references therein) and Cu complexes down-regulate nitric oxide synthase [83] it seems reasonable to suggest that these complexes may be useful in preventing these disease states. The etiology of atherosclerosis and other in¯ammatory diseases listed in the Introduction Section of this manuscript may indeed result from the formation of HOá, HOOá, and 1 O2 due to the accumulation of Oÿ 2 in relevant tissues. This accumulation of Oÿ 2 resulting from Cu de®ciency and the consequent decrease in the Cu-dependent SOD or the accumulation of H2 O2 resulting from Fe de®ciency and the consequent decrease in Fe-dependent catalase oer insights into the mechanism of disease onset and development. This then begs the question as to why small molecular mass Cu and Fe complexes, which are bioavailable forms of Cu and Fe, should be used to provide Cu and Fe for de novo synthesis of Cu2 Zn2 -SOD or catalase as well as other Cu- or Fe-dependent enzymes. Finally, with the inappropriate addition of ionically bonded Cu(II) and/or Fe(III) to biological systems there is a failure to recognize Cu(II), and/or Fe(III), catalyzed carboxylic and phosphate ester hydrolyses as well as amide hydrolysis [84,85], which are well known and do not involve HOá formation.
Acknowledgements We are indebted to Chancellor Harry P. Ward for a Special Research Grant and the Essential Metalloelement Analysis Laboratory for funding and to James Masuoka for his critical comments of our manuscript.
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