Endogenous Metabolite

Olivo et al. reported that after transurethral resection of higher-grade and higher-stage bladder tumors can produce and accumulate higher levels of Protoporphyrin IX after ALA instillation. Their findings support our results. Miyake and colleagues showed that low expression or molecular defects of ferrochelatase in urothelial cancer correlate with intracellular protoporphyrin IX accumulation. Hagiya and coworkers reported that expression levels of peptide transporter 1 and human ATP-binding cassette transporter 2 play key roles in ALA-induced protoporphyrin IX accumulation. Ogino et al. reported the stimulation of ALA-induced protoporphyrin IX accumulation in T24 cells by a ferrochelatase inhibitor and a human ATP-binding cassette transporter 2 inhibitor. Several factors seem to be involved in the mechanisms underlying the increased accumulation of Protoporphyrin IX in high-grade tumors. Which factor plays the predominant role is a subject of future research.[1]

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Mammalian porphyrins are generated from δ-aminolevulinic acid (ALA) through several consecutive enzymatic reactions to Protoporphyrin IX (PpIX), which is further complexed with an iron cation to produce heme or its derivative hemin [5].

Limited depth of penetration significantly limits photodynamic therapy of nodular basal cell carcinoma (BCC) using topical delta (5)-aminolevulinic acid (ALA). To demonstrate safety and efficacy of orally administered ALA in inducing endogenousProtoporphyrin IX (PpIX) production in BCC, 13 patients with BCC ingested ALA in a dose-escalation protocol. All dose ranges (10, 20 or 40 mg/kg single doses) resulted in formation of Protoporphyrin IX/PpIX in human skin and BCC, measurable by in vivo fluorescence spectrophotometry. The Protoporphyrin IX/PpIX fluorescence peaked in tumors before normal adjacent skin from 1 to 3 h after ALA ingestion. Gross fluorescence imaging of ex vivo specimens revealed greater PpIX fluorescence in tumor than normal skin only at the 40 mg/kg dose. Fluorescence microscopy confirmed this finding by showing distinct, full-thickness PpIX fluorescence in all subtypes of BCC only after ALA given at 40 mg/kg. Side effects were dose dependent and self limited. Photosensitivity lasting less than 24 h and nausea coinciding with peak skin PpIX fluorescence occurred at 20 and 40 mg/kg doses. After 40 mg/kg ALA, serum hepatic enzyme levels rose to a maximum within 24 h, then resolved over 1-3 weeks. Transient bilirubinuria occurred in two patients. [3]

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In this study, the biodistribution of 5-aminolaevulinic acid (ALA) and accumulation of Protoporphyrin IX (PpIX) in rats have been examined. Two groups of 21 WAG/Rij rats are given 200 mg/kg ALA orally or intravenously. Six rats serve as controls. At 1, 2, 3, 4, 6, 12 and 24 h after ALA administration, ALA and porphyrin concentrations are measured in 18 tissues and fluids. Liver enzymes and renal-function tests are measured to determine ALA toxicity. In both groups ALA concentration is highest in kidney, bladder and urine. After oral administration, high concentrations are also found in duodenal aspirate and jejunum. Mild, short-lasting elevation of creatinine is seen in both treatment groups. Porphyrins, especially Protoporphyrin IX/PpIX, accumulate mainly in duodenal aspirate, jejunum, liver and kidney (> 10 nmol/g tissue), less in oesophagus, stomach, colon, spleen, bladder, heart, lung and nerve (2-10 nmol/g tissue), and only slightly in plasma, muscle, fat, skin and brain (< 2 nmol/g tissue). In situ synthesis of porphyrins rather than enterohepatic circulation contributes to the PpIX accumulation. Confocal laser scanning microscopy shows selective porphyrin fluorescence in epithelial layers. Peak levels and total production of porphyrins are equal after oral and intravenous ALA administration.
In conclusion: administration of 200 mg/kg ALA results in accumulation of photosensitive concentrations of Protoporphyrin IX/PpIX, 1 to 6 h after ALA administration, in all tissues except muscle, fat, skin and brain. Knowledge of the time-concentration relationship should be helpful in selecting dosages, routes of administration and timing of ALA photodynamic therapy [4].

Photodynamic detection of Protoporphyrin IX in exfoliated bladder cancer cells [1]
ALA-treated and ALA-untreated urine sediments in each chamber were tested for Protoporphyrin IX fluorescence using a spectrophotometer at the appropriate settings (excitation wavelength 405 nm, emission wavelength 550–700 nm, gain 160). The gain of the spectrophotometer was adjusted when the sample intensity was out of range. The spectrum of samples treated with ALA from bladder cancer patients showed a peak at 635 nm, whereas that of ALA-untreated samples did not show such a peak (Fig. 2a). The peak was undetectable in samples from control patients (Fig. 2b).

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Evaluation of Protoporphyrin IX in exfoliated bladder cancer cells [1]
To evaluate the peak at 635 nm, the difference between the intensity of ALA-treated and ALA-untreated samples was calculated. The area under the curve, sensitivity, and specificity of this analysis were 0.68, 60%, and 65%, respectively. As mentioned, the gain was adjusted when the intensity of the sample was out of measurement range. Thus, the calculated difference was divided by the intensity of an ALA-untreated sample at 635 nm to adjust the data for different gain settings. The area under the curve, sensitivity, and specificity after adjustment were 0.74, 73%, and 63%, respectively. Some control cases that showed no peak at 635 nm were diagnosed as positive because of a difference between the sample treated and not treated with ALA; in these samples, the difference at 635 nm was detected in the range of 550 nm to 700 nm (Fig. 3b). To solve this problem, the difference between the intensity of ALA-treated and ALA-untreated samples at 600 nm was subtracted from the difference between the intensity of ALA-treated and ALA-untreated samples at 635 nm (Fig. 3a and b). After that, the adjusted difference was divided by the intensity of the ALA-untreated sample at 635 nm. This adjusted change ratio was used for the diagnosis of bladder cancer (the method of ALA-induced fluorescence cytology).

Porphyrin analysis [4]
The analysis was carried out according to Chisolm and Brown with the following modifications: the tissues were suspended in sterile water ( 1: 10 wt./vol.) and homogenized in a tissue grinder [ 2 11. To 100 ~1 of this homogenate the following were added: 700 p,l HCl 2 mmol/l and 800 p,l ethyl-acetate/glacial acetic acid (3: 1). After 10 min centrifugation at 18OOg, the ethyl-acetate phase with some protein is removed, and after a second centrifugation for 5 min at 1800g the HCl phase is measured in an LS 5B fluorescence spectrometer using a red-sensitive photomultiplier at an excitation wavelength of 410 nm and an emission wavelength of 650 nm. Although porphyrins are commonly determined by emission at 60 1 nm, under the conditions described, Protoporphyrin IX/PpIX has also a specific emission at 650 nm, which is about 80% of the emission at 601 nm and can be easily detected using a redsensitive photomultiplier. In our experience, bile acids in duodenal aspirates, and to a lesser extent other tissues, contain fluorophores with high emission at lower wavelength, which gave rise to interference in porphyrin quantitation at 601 nm. Emission at 650 nm in a direct extraction assay as described above was found to be proportional to Protoporphyrin IX/PpIX quantification, determined by a limited number of HPLC porphyrin separations. Porphyrin standards were analysed separately for their actual concentration using UV spectroscopy and the molar extinction coefficient ( .sdo7 = 0.275 L mol-’ cm-‘). Recovery of porphyrins was checked by adding standard Protoporphyrin IX/PpIX to the samples. A recovery between 90 and 100 was achieved.

This study focuses on three photosensitizers used clinically (PpIX and PF) and one photosensitizer (PPa) used in in vivo experiments. Our team proposed conjugating PPA with folic acid for photodynamic therapy (PDT) of ovarian metastases (Patent No. WO/2019/016397). By analyzing the photophysical properties of these three photosensitizers under different conditions, we found that each photosensitizer is unique, and its reaction varies significantly depending on its chemical structure and concentration. Changes in medium polarity have little effect on the UV-Vis absorption spectrum of PF, but a significant effect on PpIX and PPA. The literature generally considers PpIX to be excited at 630 nm in vitro or in vivo. This excitation wavelength is determined based on its absorption spectrum in ethanol. In aqueous media, FBS and PBS, which are closer to physiological media, its QI band is located at 641 nm. Local viscosity may vary significantly depending on the localization of the photosensitizer within the cell. We also observed that changing the solvent viscosity had little effect on the maximum absorption wavelength of QI in PpIX and PF, but for PPa, the absorption wavelength exhibited a 10 nm blue shift (from 678 nm to 668 nm). Temperature changes had minimal effect on the UV-Vis absorption spectra of PpIX and PF, but a significant effect on the UV-Vis absorption spectrum of PPa was observed in the 10–40 °C range. Finally, changing the pH value also caused a 25 nm blue shift in the QI absorption band of PPa (from 704 nm to 679 nm). Perhaps the most interesting result was the determination of the ΦΔ value in different solvents. The ΦΔ value varied significantly depending on the solvent. In toluene, we did not detect any O2, while the ΦΔ values for PpIX and PPa were 0.68 and 0.49, respectively, which was quite good. In ethanol, the ΦΔ values for PpIX, PPa, and PF were 0.92, 0.53, and 0.80, respectively. If D2O is used instead, the 1O2 of PpIX or PPa cannot be detected, and the ΦΔ value of PF drops to 0.15. In addition, in practical applications, photosensitizers are ideally located in the cellular environment. The presence of proteins, lipids and other biomolecules can also affect the photophysical properties of photosensitizers. This raises the question of what type of experiment should be used and what solvent should be used in solution when conducting in vitro studies. [2]

[1]. Protoporphyrin IX induced by 5-aminolevulinic acid in bladder cancer cells in voided urine can be extracorporeally quantified using a spectrophotometer. Photodiagnosis Photodyn Ther. 2015 Jun;12(2):282-8.

[2]. Photophysical Properties of Protoporphyrin IX, Pyropheophorbide-a and Photofrin® in Different Conditions. Pharmaceuticals (Basel). 2021 Feb 9;14(2):138.

[3]. Protoporphyrin IX fluorescence induced in basal cell carcinoma by oral delta-aminolevulinic acid[J]. Photochem Photobiol. 1998 Feb;67(2):249-55.

[4]. 5-Aminolaevulinic acid-induced protoporphyrin IX accumulation in tissues: pharmacokinetics after oral or intravenous administration[J]. J Photochem Photobiol B. 1998 Jun 15;44(1):29-38.

[5]. G-quadruplexes sense natural porphyrin metabolites for regulation of gene transcription and chromatin landscapes. Genome Biol. 2022 Dec 15;23(1):259.

Protoporphyrin is a cyclic tetrapyrrole composed of a porphyrin ring with four methyl groups at positions 3, 8, 13, and 17, two vinyl groups at positions 7 and 12, and two 2-carboxyethyl groups at positions 2 and 18. It is the parent compound of protoporphyrins. It can be used as a photosensitizer, metabolite, E. coli metabolite, and mouse metabolite. It is the conjugate acid of protoporphyrin acid and protoporphyrin(2-). Protoporphyrin IX is a metabolite found in or produced by E. coli (K12 strain, MG1655 strain). It has also been reported to have been found in Homo sapiens and thrushes, with relevant data available. Protoporphyrin IX is a tetrapyrrole containing four methyl groups, two propionic acids, and two vinyl side chains; it is a metabolic precursor of heme, cytochrome c, and chlorophyll. Protoporphyrin IX is generated by the oxidation of protoporphyrinogen methylene bridges catalyzed by protoporphyrinogen oxidase. Protoporphyrin IX is a metabolite found or produced in Saccharomyces cerevisiae. Background: We evaluated the feasibility of photodynamic diagnosis of bladder cancer by spectrophotometric analysis of urine samples treated with 5-aminolevulinic acid (ALA) in vitro. Methods: We recruited 61 patients with histologically confirmed bladder cancer following transurethral resection of bladder tumors as the bladder cancer group and 50 outpatients without a history of urothelial carcinoma or cancer-related manifestations as the control group. Half of the urine samples were incubated with ALA (ALA-treated group), and the other half were untreated (ALA-untreated group). To detect intracellular protoporphyrin IX levels, we measured the absorbance of the samples at an excitation wavelength of 405 nm using a spectrophotometer. The absorbance difference at 635 nm was calculated between the ALA-treated and untreated samples. Results: The absorbance difference in the bladder cancer group was significantly higher than that in the control group (p < 0.001). The absorbance difference in patients with high-grade tumors was also significantly higher than that in patients with low-grade tumors (p = 0.004), and the absorbance difference in patients with invasive bladder cancer was also significantly higher than that in patients with non-invasive bladder cancer (p = 0.007). The area under the curve was 0.84. The sensitivity and specificity of the method were 82% and 80%, respectively. Conclusion: We demonstrate that the level of protoporphyrin IX in urinary cells after ALA treatment can be quantitatively detected using a spectrophotometer in patients with bladder cancer. Therefore, this cancer detection system has potential for clinical application. [1] Photodynamic therapy (PDT) is an innovative method for treating malignant or diseased tissues. The efficacy of PDT depends on the light dose, oxygen supply, and the nature of the photosensitizer (PS). Depending on the medium, the photophysical properties of PS can change, leading to an increase or decrease in fluorescence emission and the generation of reactive oxygen species (ROS), especially singlet oxygen (1O2). In this study, UV-Vis absorption spectroscopy, fluorescence emission spectroscopy, singlet oxygen emission spectroscopy and time-resolved fluorescence spectroscopy were used to investigate the effects of solvent polarity, viscosity, concentration, temperature and pH on the photophysical properties of protoporphyrin IX, pyrophyllite a and Photofrin®. [2]

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Background: G-quadruplexes (G4) are a unique non-classical nucleic acid secondary structure that is believed to be able to physically interact with transcription factors and chromatin remodeling factors, thereby regulating cell type-specific transcriptomes and shaping chromatin structure.
Results: Based on the direct interaction between G4 and natural porphyrins, we established a genome-wide approach to analyze the binding sites of iron-coordinated porphyrin heme in chromatin. Heme promotes the formation of G4 across the genome, inhibits transcription initiation and alters chromatin structure, including reducing the levels of H3K27ac and H3K4me3 modifications in promoter regions. Interestingly, the G4 state does not participate in the classic heme-BACH1-NRF2-mediated enhancer activation process, revealing an unprecedented G4-dependent transcriptional metabolic regulation mechanism. In addition, heme treatment can induce specific gene expression profiles in hepatocytes, highlighting the potential of porphyrins to regulate gene transcription in vivo. Conclusion: These studies demonstrate that G4 functions as a sensor of natural porphyrin metabolites in cells, revealing a G4-dependent mechanism for gene transcription and chromatin structure metabolism regulation, which will deepen our understanding of G4 biology and the contribution of cellular metabolites to gene regulation. [5]

C34H34N4O4

562.65816

562.257

C, 72.58; H, 6.09; N, 9.96; O, 11.37

553-12-8

50865-01-5 (di-hydrochloride salt)

4971

Brown to black solid powder

1.3±0.1 g/cm3

1122.0±65.0 °C at 760 mmHg

632.4±34.3 °C

0.0±0.3 mmHg at 25°C

1.674

7.33

4

6

8

42

1010

0

O=C(O)CCC1=C2/C=C3C(CCC(O)=O)=C(C)C(/C=C(N/4)/C(C)=C(C=C)C4=C\C5=N/C(C(C=C)=C5C)=C\C(N2)=C1C)=N/3

ZCFFYALKHPIRKJ-UHFFFAOYSA-N

InChI=1S/C34H34N4O4/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/h7-8,13-16,35-36H,1-2,9-12H2,3-6H3,(H,39,40)(H,41,42)

3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethyl-22,23-dihydroporphyrin-2-yl]propanoic acid

protoporphyrin IX; protoporphyrin; 553-12-8; Ooporphyrin; Protoporpyrin IX; Porphyrinogen IX; Kammerer's prophyrin; Protoporphyrin IX (VAN);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO : ~2 mg/mL (~3.55 mM)
Ethanol :< 1 mg/mL

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.)