Serum albumin leads to false-positive results in the XTT and the MTT assay

Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.     

Assessment of Cell Viability in Intact Glandular Tissue in <Emphasis Type="Italic">Chironomus ramosus</Emphasis> using Dye-exclusion and Colorimetric Assays

Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r² = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future.     

Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction by mycoplasmas.

We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.     

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.     

Efficiency of Neutral Red,Evans Blue and MTT to assess viability of the freshwater microalgae Desmodesmus communis and Pediastrum boryanum

This study assessed the efficacy of three cell viability assays – Methyl‐thiazolyl‐tetrazolium (MTT), Evans Blue, and Neutral Red – for two freshwater microalgae species, Desmodesmus communis and Pediastrum boryanum (Chlorophyceae), in order to find suitable techniques to detect the levels of pollution in water ecosystems. Following exposure to a glyphosate‐based herbicide, our results showed that Evans Blue did not adequately measure cell viability in either species, while MTT and Neutral Red were able to detect decreased cellular viability for both algae in response to herbicide exposure. Overall, however, Neutral Red proved to be more sensitive than MTT for these algae.     

Investigation of different modifications to the tetrazolium based colorimetric viability assay

Summary Three commonly used solvents for the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based viability assay for mammalian cells were compared: Acid/propan-2-ol, dimethyl sulfoxide (DMSO) and a lysing buffer containing sodium dodecylsulphate (SDS). Acid/propan-2-ol and DMSO could only be used when most of the medium was removed from the cell suspensions, whereas the lysing buffer was found to perform satisfactorily for both hybridomas and fibroblast cell lines without medium removal for cell densities up to 10⁶ cells mL⁻¹. Furthermore a newly synthesized tetrazolium salt was investigated, which forms a water soluble formazan upon reduction and thus eliminates the need for a solvent. However this salt adds a new complication to the method: the need for an electron carrier. For this reason we do not find that the new tetrazolium salt has any practical advantages over MTT.     

Effect of aluminum and lead salts on lipid peroxidation and cell survival in human skin fibroblasts

The aim of this study was to see whether aluminum (Al) and lead (Pb) salts are toxic for cultured human fibroblasts under different experimental conditions, in the controllable situation offered by cell cultures. Cell survival and membrane lipid peroxidation served as markers of Al and Pb toxicity. Evaluation of the living cells was carried out using a colorimetric method, the mitochondrial reduction of 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Lipoperoxidation assay was performed on whole cell homogenates by measuring thiobarbituric acid-reactive substances (TBARS) produced after incubation with ascorbic acid-ferrous sulfate. Al(III) and Pb(II) salts (300 μM) produce a considerable decrease in cell survival after an exposure period of 4 d, evident with the three fetal calf serum concentrations in the culture media: 2, 5, and 10%. Taking into account in vitro cell aging, the cytotoxic effects of Al(III) and Pb(II) are greater in senescent fibroblasts than in young cells. Lead-induced cytotoxicity is higher than Al-induced cytotoxicity. A mechanism that contributes to cellular toxicity is membrane lipid peroxidation; our results demonstrate that Al(III) and Pb(II) ions, 400 μM, exert an antioxidant-like effect or a pro-oxidant action on cell membranes depending on exposure time. We describe significant increases in TBARS formation associated with the presence of 400 μM Al(III) or Pb(II) salts in the culture media. Our study also revealed that these heavy metals induce a cell age-dependent action on membrane lipoperoxidation that is greater in senescent fibroblasts and this could have severe consequences for maintenance of cellular integrity.     

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider line arrange, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4°C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative,whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability

Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.     

The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent

The water-soluble tetrazolium salt (WST-1) assay is frequently used to assess cell proliferation. However, our study showed that in normal and cancerous keratinocytes, this assay is more responsive to changes in oxygenation than to rates of cell growth. Stimulation of keratinocyte proliferation by low Ca²⁺ and suppression of proliferation by nocodazole resulted in modest changes in WST-1 readings, whereas gradually reducing the level of oxygen in the cellular environment from ambient (21%) to near anoxic (0.1%) revealed a very strong negative correlation between cell oxygenation and WST-1 reagent reduction. In contrast, the very similar MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, which uses a different tetrazolium salt, showed no sensitivity to the level of oxygen. Unlike MTT, WST-1 reagent is reduced extracellularly through trans-plasma membrane transport (tPMET), thereby suggesting that tPMET is oxygen dependent. We propose that the WST-1 assay can be developed into a sensitive quantitative method to evaluate cell oxygenation in vitro and used to study the role of hypoxia and tPMET in homeostasis and disease (e.g., cancer). At the same time, WST-1 assay should be used cautiously to assess cell viability or proliferation because readings can be affected by certain extrinsic (low atmospheric oxygen or high density culture) or intrinsic (defects in oxygen-sensing pathways) factors.     

Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 μg/mL of oleuropein or 50 μg/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G₁ to S phase transition manifested by the increase of cell number in G₀/G₁ phase.     

Effects of high glucose concentration on survival and respiration of human endothelial cells.

To determine whether or not endothelial cell survival was decreased after incubation with high glucose concentrations in culture media, we studied the influence of D-glucose or L-glucose (a non-metabolizable stereoisomer of D-glucose) on cell survival using the trypan-blue exclusion test. Simultaneously, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to measure both the mitochondrial respiratory chain activity and cell viability. Respiratory chain activity per cell increased when D-glucose concentrations rose but at the same time trypan-blue excluded cells were decreased. Comparison with data in the literature showed that the MTT assay was not reliable for studies involving endothelial cell survival when glucose reduction was affected on these cells. It seems important to check MTT assay reliability carefully when it is used for drugs affecting glucose metabolism, or with other cell types.     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

The colorimetric assay previously described by Mosmann for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.     

In vitro cytotoxicity of the organophosphorus insecticide methylparathion to FG-9307, the gill cell line of flounder (Paralichthys olivaceus)

FG-9307, a cell line derived from the gill of flounder Paralichthys olivaceus, was used to determine the acute cytotoxic effects of the organophosphorus insecticide methylparathion. The cytotoxic effects of methylparathion were initially measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay, and cell protein assay. Results indicated that concentrations of methylparathion ranging from 5 μg/ml to 60 μg/ml were toxic, and there was no significant difference in cytotoxic effects between the three test systems. Thus, the FG-9307 cell line is one of several choices for evaluating the acute toxicities of organophosphorus insecticides such as methylparathion. The ultrastructure of the cells was also studied. It was found that the ultrastructure of the cells was markedly altered by methylparathion, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear necrosis, and production of numerous lysosomes and lipid vacuoles. This appears to be the first report that a marine fish cell line can be used for acute in vitro cytotoxicity evaluation of methylparathion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids

Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic β-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on β-cell survival and metabolism. We treated the rat β-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F β-cells. However, ethanol causes β-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human β-cells.     

Reduction of tetrazolium salts catalyzed by soluble diaphorase in rat liver and embryos of Vicia faba

Summary The presence of two diaphorases has been shown in rat liver and embryos of Vicia fdba. One of these, the NAD(P)H tetrazolium reductase, was firmly bound in the section and was not lost into the incubation medium under conditions of histochemical assay The second diaphorase (soluble diaphorase) was lost from the section into the incubation medium during the first five minutes of incubation. This soluble diaphorase from both rat liver and embryos of V. faba is capable of transferring electrons from NAD(P)H to MTT, INT, NBT and TNBT, but not to tellurite, TTC, BT and NT. The behaviour of the soluble diaphorase in histochemical reactions involving tetrazolium salts as electron acceptors is discussed.     

Estradiol attenuation of β-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17β-estradiol (βE2) has been shown to attenuate the toxicity of β-amyloid peptides (Aβ) in neuronal cultures with the effective concentration of βE2 ranging from low nM to high μM. This study compares the effective neuroprotective concentration of βE2 against both Aβ-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective βE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum βE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM βE2 conferring significant protection in the calcein AM assay but 1 μM βE2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic Aβ concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM βE2 against H_{2 2} toxicity. These results indicate that low concentrations of βE2 can attenuate Aβ and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of βE2-mediated attenuation of Aβ toxicity.     

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

The use of tetrazolium salts in the histochemical demonstration of succinic dehydrogenase activity in plant tissues

Summary Succinic dehydrogenase activity was determined in fresh or cryostat sections of tissues from Allium cepa, Vicia faba, Pisum sativum and Helianthus tuberosus using different tetrazolium salts as electron accepters. In 10 fresh or frozen sections a reaction was obtained with TNBT, NBT, MTT, and INT but not with NT, BT or TTC. In contrast a reaction was obtained with each of the tetrazolium salts in 50–150 fresh or frozen sections. The observed differences in the abilities of the tetrazolium salts to demonstrate succinic dehydrogenase activity are discussed.The sites of acceptance of electrons from the electron transport pathway in plant cells by the tetrazolium salts has been demonstrated cytochemically, and shown to differ from those observed in animal cells in that unlike animal cells, there is no apparent acceptance of electrons by MTT and INT from cytochrome C₁-C region of the pathway.