Biochemical; metalloporphyrin

Gossypol prevents the liberation of oxygen from oxyhemoglobin and exerts a hemolytic effect on erythrocytes. In excessive dosages of gossypol, an extreme burden is placed upon the respiratory and circulatory organs owing to the reduced oxygen carrying capacity of blood. Chromium protoporphyrin (CrPP) has been shown to either competitively suppress or to significantly ameliorate a variety of naturally occurring or experimentally induced forms of jaundice in animals and man. In this communication, a novel tissue dependent response to gossypol (50 micromol/kg bw) and gossypol in association with CrPP (50 micromol/kg bw) is described. Our results revealed that gossypol stimulated the hepatic, splenic, and renal delta-aminolevulinic acid synthase (ALA-S) activity, the heme biosynthetic enzyme, and simultaneous administration of CrPP and gossypol synergized the gossypol-mediated increase of ALA-S activity. Gossypol was found to be a potent stimulator of heme oxygenase (HMOX) activity in rat liver and kidney to varying degrees. This tissue response contrasted with that of the spleen, where gossypol decreased the activity of the enzyme. In consonance with the increased hepatic and renal HMOX activity, a marked increase was observed in total serum bilirubin concentration in gossypol treated rats. When rats were given CrPP simultaneously with gossypol, the gossypol mediated increase in hepatic and renal HMOX activity was effectively blocked. Furthermore, the increase in enzymatic activity was accomplished by a decline in the total microsomal protein content on gossypol administration. These findings emphasize the toxic effect of gossypol in eliciting increased heme degradation by stimulating HMOX activity in the liver and the kidney and the potential usefulness of CrPP in experimental and perhaps clinical conditions in which hyperbilirubinemia occurs [1].

Experimental Animals [1]
Male Wistar Rats of weight range 150–200 g from our laboratory maintained colony were used as experimental models in the investigation. Only healthy animals were taken in individual cages having raised wire mesh floors. The animals were kept on fasting for 20 h but had free access to water. After 20 h the animals were divided into four groups with eight animals per group.

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Animal Treatment [1]
Group I: Animals of this group were treated as control and were administered equivalent amount of saline subcutaneously.
Group II: 50 µmol/kg bw of gossypol was given subcutaneously to animals in this group.
Group III: Animals in this group were administered 50 µmol/kg bw of CrPP subcutaneously.
Group IV: Animals in this group were given 50 µmol/kg bw of gossypol along with 50 µmol/kg bw of CrPP subcutaneously.
The solutions of gossypol and CrPP for administration were prepared fresh in small volumes, in dark, because of their photosensitivity and unstable nature. Metalloporphyrins require an alkaline media for dissolving i.e., for making 1 mL solution, the porphyrin was dissolved in 0.2 mL of 0.02 N NaOH and the volume was then made up by potassium phosphate buffer (pH 7.4). Stock solution of gossypol was prepared in 95% ethanol. The gossypol concentration was determined by measuring absorbance at 372 nm and using a value of € = 1.48 × 104 L mol−1cm−1 (Finaly et al., Citation[[1993]]).

[1]. Effect of gossypol in association with chromium protoporphyrin on heme metabolic enzymes. Artif Cells Blood Substit Immobil Biotechnol. 2004 Feb;32(1):159-72.
[2]. Protoporphyrin IX: the Good, the Bad, and the Ugly. J Pharmacol Exp Ther. 2016;356(2):267-275.

These studies reveal an important concept: ALA-S activity is regulated by tissue specificity. The biochemical basis of the observed differences in enzyme regulation can be explained, at least in part, by the physiological characteristics of these tissues. We propose that heme synthesis in hepatocytes must respond to external stimuli because the detoxification or metabolism of exogenous substances and endogenous steroids requires the synthesis of heme proteins, such as cytochrome P-450. Under our experimental conditions, we observed that gossypol increased ALA-S activity in the liver, spleen, and kidneys. We found that the combined action of gossypol and CrPP further increased intracellular heme concentration, meaning that combined administration significantly enhanced ALA-S activity. Studies have shown that intracellular heme synthesis is competitive, involving the synthesis of multiple heme proteins, including microsomal cytochrome P-450 and b5, mitochondrial cytochrome catalase, and tryptophan pyrrolase. The remaining heme not used for apolipoprotein synthesis is either used to promote ALA-S synthesis (thus enhancing net heme synthesis) or metabolized into bile pigments by heme oxygenase. We observed that ALA-S activity was stimulated in tissues under our experimental conditions. The mechanism by which exogenous substances induce increased ALA-S activity is not yet clear. Inducers may act directly on genes, increasing their transcription rate. Gossypol may increase the activity of this enzyme by reducing heme concentration. Gossypol may induce de novo synthesis of ALA-S rather than activating existing enzyme systems. Given the broad-spectrum activity of gossypol, the reported stimulatory effect of gossypol on HMOX activity, which is significantly attenuated when co-administered with CrPP, may have important biological significance. Gossypol significantly increased HMOX activity in the liver and kidneys of tested rats. This finding suggests that the biological basis of gossypol-induced hyperbilirubinemia may be primarily related to the accelerated rate of heme protein conversion to bilirubin. The cellular mechanisms underlying gossypol-mediated increases in HMOX activity remain unclear, but may involve: (a) the intermediate role of hemoglobin released during hemolysis; (b) the direct action of the parent compound; and (c) the activity of gossypol's active metabolites. However, this study could not select for any of these possibilities. Therefore, the biochemical effects behind this observation remain unknown. Given gossypol's hemolytic effect on erythrocytes, it is evident that hemoglobin released during erythrocyte hemolysis may stimulate microsomal HMOX activity. This, in turn, could significantly lead to elevated serum bilirubin levels. These concepts are consistent with findings that hemoglobin infusion induces HMOX activity in rat kidneys (Pimstone et al., 1971); methemoglobin promotes increased HMOX activity in rat livers (Tenhunen et al., 1970); and postpartum hyperbilirubinemia is associated with increased HMOX activity in the liver and kidneys of rats (Maines and Kappas, 1978b). Similarly, the possibility that gossypol and/or its metabolites directly induce HMOX activity cannot be ruled out. CrPP can effectively inhibit gossypol-mediated increases in HMOX activity, indicating that this metalloporphyrin can be used to inhibit enzyme activity under various hemolytic conditions. Gossypol-mediated induction of hepatic and renal HMOX activity is accompanied by hyperbilirubinemia. Therefore, we can speculate that, provided that the exogenous substances are delivered to the target organ in sufficient quantities, their ability to act as enzyme stimulators or inhibitors may depend on their metabolic status in tissue cells. The effect on HMOX activity can be explored from the extent to which a specific tissue maintains its metabolism after being subjected to such changes. [1]

C34H32CLCRN4O4

648.1

647.152

C, 63.01; H, 4.98; Cl, 5.47; Cr, 8.02; N, 8.64; O, 9.87

41628-83-5

120389-54-0

134129038

Typically exists as solid at room temperature

1.998

2

8

8

44

1020

0

C=CC1=C2C=C3C(C)=C(CCC(=O)O)C4=CC5=NC(=CC6=NC(=CC(=C1C)N2[Cr](Cl)N34)C(C=C)=C6C)C(C)=C5CCC(=O)O CC1C(C=C)=C2C=C3C(C)=C(CCC(O)=O)C4=CC5=NC(=CC6=NC(=CC=1N2[Cr](Cl)N43)C(C=C)=C6C)C(C)=C5CCC(O)=O |c:20,24,t:16|CopyCopied

KGPQQEWHGUXPBK-UHFFFAOYSA-K

InChI=1S/C34H34N4O4.ClH.Cr/c1-7-21-17(3)25-13-26-19(5)23(9-11-33(39)40)31(37-26)16-32-24(10-12-34(41)42)20(6)28(38-32)15-30-22(8-2)18(4)27(36-30)14-29(21)35-25;;/h7-8,13-16H,1-2,9-12H2,3-6H3,(H4,35,36,37,38,39,40,41,42);1H;/q;;+3/p-3

3-[18-(2-carboxyethyl)-7,12-bis(ethenyl)-3,8,13,17-tetramethylporphyrin-21,22-diid-2-yl]propanoic acid;chlorochromium(2+)

Cr(III) Protoporphyrin IX Chloride; 41628-83-5; 3-[18-(2-carboxyethyl)-7,12-bis(ethenyl)-3,8,13,17-tetramethylporphyrin-21,22-diid-2-yl]propanoic acid;chlorochromium(2+); Cr(III)ProtoporphyrinIXChloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)