Abstract

Protoporphyrin IX (PPIX) is formed from δ-aminolevulinic acid (ALA) during heme biosynthesis. Due to its cyclic tetrapyrrole core structure, it absorbs in the visible region of the electromagnetic spectrum and is thus colored. Both ALA and PPIX have become of great interest to neurosurgery, because in high-grade glioma, ALA diffuses into the tumor and is converted to PPIX. Fluorescence-guided resection (FGR) takes advantage of both the enrichment of PPIX in the tumor and its fluorescent properties, which enable visualization of tumor tissue. ALA-mediated FGR thus maximizes the extent of resection with better prognosis for patients. Tumor cells are able to produce porphyrins naturally or after administration of ALA, which is also reflected in elevated plasma fluorescence of cancer patients. PPIX might thus serve as a biomarker for monitoring of the tumor burden. A liquid chromatography-mass spectrometry (LC-MS)-based method is presented to quantify PPIX in blood and serum in the context of current fluorescence-based diagnostics. The method is able to distinguish between zinc PPIX, a component of red blood cells of importance in the detection of lead poisoning and iron deficiency anemia, and metal-free PPIX. In a proof-of-principle study, it was used to follow a time course of a glioblastoma patient undergoing surgery and confirmed elevated PPIX blood levels before ALA administration. During surgery, these blood levels increased about four-fold. The here developed 10 min reversed-phase LC-target MS method now allows patient screening with high specificity and throughput.

Highlights

  • Cyclic tetrapyrroles are biologically important molecules as they form the core structure of prosthetic groups such as porphyrins, natural pigments like chlorophyll, and of vitamers

  • ZnPPIX is the predominant species of Erythrocyte protoporphyrin (EP) in circulating erythrocytes while free protoporphyrin IX (PPIX) is only present in trace amounts [20, 30, 31]; the specific quantification of metal-free PPIX next to ZnPPIX is challenging

  • For the detection of PPIX in whole blood or serum, we developed an high-performance liquid chromatography (HPLC)-MS method using an HP1100 HPLC (Agilent, Waldbronn, Germany) coupled to an Esquire 3000 ion trap mass spectrometer (Bruker Corp., Bremen, Germany)

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Summary

Introduction

Cyclic tetrapyrroles are biologically important molecules as they form the core structure of prosthetic groups such as porphyrins (e.g. heme), natural pigments like chlorophyll, and of vitamers (cobalamins, e.g. vitamin B12). During heme synthesis from glycine and succinyl-CoA (Figure 2) a number of intermediates including δ-aminolevulinic acid (ALA) are produced until, protoporphyrin IX (PPIX) is converted to heme by insertion of a divalent iron (Fe (II), catalyzed by ferrochelatase) [1]. Both ALA and PPIX have become of great interest to neurosurgery, because in gliomas, ALA diffuses into the tumor and induces PPIX-synthesis [6]. ALA-FGR maximizes the extent of tumor resection with improved prognosis for patients. We have used liquid chromatography coupled to mass spectrometry (LC-MS) to quantify PPIX in blood and serum and describe it in the context of current practice in PPIX diagnostics below

Erythrocyte protoporphyrin analysis
Spectrophotometry and –fluorometry
Liquid chromatography
Protoporphyrin IX: a potential biomarker for cancer screening
PPIX quantification
Sample preparation
Gas phase fragmentation
Measurement of clinical samples
Findings
Conclusion
Full Text
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