**1. 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). Due to their large conjugated systems, they absorb in the visible region of the electromagnetic spectrum and are thus colored [1, 2].

Porphyrins occur naturally as metal complexes with the red pigment in blood cells, heme, being the best-known example. Hemes (**Figure 1**), the cofactors of

#### **Figure 1.**

*Porphyrin structures. Porphin (left), the simplest representative, and heme b (right), the prosthetic group of, e.g., hemoglobin, myoglobin, catalase, and cytochromeP450. Other members of the heme group (e.g. heme c in cytochrome C) differ slightly in the side chains. Porphyrins chelate divalent ions such as iron in heme. Due to their delocalized system of* π*-electrons they fluoresce after excitation. Different nitrogen forms in the pyrrole ring are labelled (red/italic, blue/bold).*

hemoproteins (e.g. hemoglobin, myoglobin) [3, 4], are complexed to iron and occur ubiquitously. They are critical to life, because hemoproteins are involved in the transport of diatomic gases (respiration), chemical catalysis and electron transfer [5].

During heme synthesis from glycine and succinyl-CoA (**Figure 2**) a number of intermediates including δ-aminolevulinic acid (ALA) are produced until, ultimately, 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]. A surgical method has been developed taking advantage of both the enrichment of PPIX in the tumor

#### **Figure 2.**

*Heme synthesis from succinyl-CoA to PPIX and ultimately heme. Synthesis of ALA is the rate-limiting step and under negative feedback control of heme (green/ dashed). A deficiency of iron supply limits heme synthesis and leads to PPIX accumulation. Zinc can then substitute for iron and ferrochelatase catalyzes the formation of zinc PPIX (red/dotted) [1].*

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*Protoporphyrin IX Analysis from Blood and Serum in the Context of Neurosurgery…*

of blood from cancer patients differ from those of control subjects [8–11].

and its fluorescent properties, which enable visualization of the tumor area (fluorescence-guided resection (FGR)) [7]. Alternating between normal white light illumination and violet-blue excitation light, the vital tumor tissue can be identified by its red/ pink color during surgery; the distinction of infiltration zone and healthy brain tissue is improved. ALA-FGR thus maximizes the extent of tumor resection with improved prognosis for patients. In this context, the question arose whether or not PPIX blood levels could be used to monitor tumor regrowth, because the spectral characteristics

The use of a simple analytic procedure would be cost-effective at lower strain for the patient. We have thus used liquid chromatography coupled to mass spectrometry (LC-MS) to quantify PPIX in blood and serum and describe it in the context of

Erythrocyte protoporphyrin (EP) served as a diagnostic marker for lead poisoning and environmental lead pollution as well as for iron deficiency anemia at the end of the 20th century [12]. From 1972 to 1991, it was officially recommended as the primary screening test for childhood lead poisoning by the Center for Disease Control and Prevention in the United States [13–15]. For the clinical diagnosis of porphyrias [16], rare disorders resulting from enzyme variability in heme biosynthesis, the porphyrin pattern is determined in blood, urine and faeces based on

Taking advantage of the strong absorption of porphyrins in the Soret band (380– 430 nm) and their fluorescence, spectrophotometric and -fluorometric methods have been preferred for EP determination so far. The free erythrocyte porphyrin (FEP) test [17, 18], was, however, based on liquid-liquid extraction (LLE) at acidic pH, which dissociated zinc protoporphyrin (ZnPPIX) to metal-free PPIX during the extraction process. Thus, a sum parameter with different - and unknown - contributions of free PPIX and ZnPPIX was measured leading to false conclusions. The ratio of ZnPPIX to metal-free PPIX in erythrocytes varies, because in lead poisoning and iron deficiency anemia, ZnPPIX is accumulated in the blood,

Hence, neutral ZnPPIX-specific LLE methods were developed, but they suffered from poor extraction efficiency [20, 21]. The widely applied ethyl acetate-acetic acid LLE method had three problems [17, 18]: First, the low extraction efficiency of PPIX from whole blood in comparison to the extraction from pre-diluted blood, which provided better precision of analysis; second, impurities in ethyl acetate influencing fluorescence and requiring pre-tests of reagent batches; third, the instability of PPIX standards prepared with deionised water. Thus, EP analysis required great attention to detail, because method modification, sample contamination or aging of standards and reagents had a great impact on analysis [22]. As a result, inter-laboratory comparison of EP results was generally poor while intra-laboratory precision was good [23].

In 1977, a hematofluorometer (HF) was designed for the detection of ZnPPIX in a drop of whole blood without sample pretreatment, which allowed immediate, simple and inexpensive detection [24]. Spectrophotometric and -fluorometric analysis became conventional analytical practice, but the inter-laboratory

whereas in protoporphyria, the metal-free PPIX is elevated [12, 19, 20].

*DOI: http://dx.doi.org/10.5772/intechopen.95042*

current practice in PPIX diagnostics below.

**2.1 Erythrocyte protoporphyrin analysis**

**2.2 Spectrophotometry and –fluorometry**

**2. Protoporphyrin IX detection**

fluorescence techniques.

*Protoporphyrin IX Analysis from Blood and Serum in the Context of Neurosurgery… DOI: http://dx.doi.org/10.5772/intechopen.95042*

and its fluorescent properties, which enable visualization of the tumor area (fluorescence-guided resection (FGR)) [7]. Alternating between normal white light illumination and violet-blue excitation light, the vital tumor tissue can be identified by its red/ pink color during surgery; the distinction of infiltration zone and healthy brain tissue is improved. ALA-FGR thus maximizes the extent of tumor resection with improved prognosis for patients. In this context, the question arose whether or not PPIX blood levels could be used to monitor tumor regrowth, because the spectral characteristics of blood from cancer patients differ from those of control subjects [8–11].

The use of a simple analytic procedure would be cost-effective at lower strain for the patient. We have thus 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.
