The erythrocyte protoporphyrin extraction method using acetone, which allows for the simultaneous analysis of FPP and ZnPP, was first described in 1981; however, it did not gain widespread popularity²⁹. The authors originally used an excitation wavelength of 420 nm, resulting in a higher representation of ZnPP in the emission spectrum. Nevertheless, selecting a 405 nm excitation wavelength in our protocols was driven by the need to reduce spectral interference caused by bilirubin’s strong absorption. This is particularly relevant in our study, where patients with liver diseases and other comorbidities often have elevated bilirubin levels. The widespread adoption of hematofluorometers, such as systems based on direct fluorescence analysis of protoporphyrins from blood drops using the front-face method, further limited the application of the acetone extraction technique. This equipment is specifically optimized for ZnPP quantification, utilizing excitation wavelengths of 420–430 nm and detecting fluorescence emission at 594 nm, corresponding to the characteristic peak of ZnPP. However, FPP does not fluoresce at these wavelengths and is, therefore, not detected by this method.

Our study demonstrated that, under 405 nm excitation, fluorescence at 632 nm from erythrocyte extracts originates predominantly from FPP, as confirmed by spectral decomposition. The prognostic value of fluorescence-based markers indicates that FPP and direct fluorescence intensity at 632 nm reliably reflect mortality risk in patients with CAP. Kaplan–Meier survival analyses further support their clinical relevance, underscoring their potential as reliable indicators of adverse outcomes. Importantly, these associations remained statistically significant after adjustment for age and the CCI in multivariable logistic regression models, highlighting their independent prognostic value. In contrast, although ZnPP also demonstrated a statistical association with mortality, its predictive capacity was weaker, suggesting a more limited role in risk stratification. Direct fluorescence at 632 nm offers a practical alternative to full spectral deconvolution, providing a simpler yet comparably robust approach suitable for translational and clinical applications. Moreover, strong correlations between F632/FPP and key clinical and laboratory parameters reinforce their potential utility as integrative markers of disease severity, systemic inflammation, and metabolic disturbances.

The elevated mortality observed in patients with high FPP fluorescence likely reflects the convergence of several pathophysiological mechanisms associated with pneumonia. Possible contributors may include inflammation-related iron dysregulation with impaired heme synthesis, hypoxia-driven erythropoiesis, oxidative stress, bacterial iron sequestration, and zinc deficiency. Nevertheless, no prior study has explicitly identified elevated erythrocyte FPP as a biomarker of pneumonia-related mortality. Consequently, these mechanistic considerations remain largely theoretical, highlighting the need for further research.

Among these factors, the most critical is the activation of the interleukin-6 (IL-6)–hepcidin axis during the acute phase of pneumonia, which profoundly disrupts iron metabolism and impairs heme synthesis^30,31. Under normal physiological conditions, iron is delivered to erythroblast mitochondria via the transferrin–transferrin receptor 1 complex. In the mitochondrion, ferrochelatase catalyzes the insertion of ferrous iron insertion into protoporphyrin IX, yielding heme. The newly synthesized heme is subsequently exported to the cytoplasm, where it binds to globin to form functional hemoglobin³². When pathogens invade the lungs, alveolar macrophages recognize bacterial or viral components via pattern recognition receptors (e.g., Toll-like receptors), activating the NF‑κB and STAT3 pathways³³. This, in turn, triggers a pronounced increase in IL‑6 production, driving hepatocytes and local immune cells to produce hepcidin³⁴. Hepcidin then binds to ferroportin, inducing its internalization and degradation, thereby halting iron release from macrophages and enterocytes. Reduced iron availability limits the metal’s transport into erythroblast mitochondria and impedes its incorporation into protoporphyrin IX. Consequently, erythroid maturation is disrupted, leading to elevated levels of FPP and ZnPP.

It is important to note that pneumonia, particularly in severe clinical presentations, is often associated with significant hypoxia. The organism attempts to enhance erythropoiesis by increasing erythropoietin (EPO) production, which drives the proliferation of erythroid precursors in the bone marrow³⁵. This occurs through stabilizing hypoxia-inducible factor alpha (HIF-α), which, due to inhibited degradation by prolyl hydroxylase, accumulates and translocates to the nucleus, activating EPO transcription in renal peritubular cells. Increased EPO stimulates erythroid progenitors via JAK2-STAT5 signaling, enhancing red blood cell production³⁶. HIFs also promote the expression of iron-regulatory protein genes, increasing intracellular iron availability essential for heme biosynthesis³⁷. Concurrently, erythroferrone (ERFE) is secreted by erythroblasts in response to elevated EPO levels, suppressing hepcidin expression and enhancing iron mobilization from storage sites to support erythropoiesis³⁸. The net result is a dynamic interplay: while infection and inflammation elevate hepcidin to restrict bacterial access to iron, hypoxia and erythropoiesis-associated signals, including ERFE, attempt to suppress hepcidin and secure adequate iron for hemoglobin synthesis. Animal studies have shown that pneumonia increases hepcidin expression, dependent on IL-6 signaling³⁰. Similarly, patients with pneumonia display elevated hepcidin levels that correlate with systemic inflammation markers such as IL-6 and C-reactive protein^39,40. Schoorl et al. reported that hepcidin-25 levels peaked at hospital admission in patients with CAP and declined by approximately 50% by day 4 and 75% by day 14⁴¹. Likewise, Aiyro et al. documented a 6.3-fold reduction in hepcidin levels over a six-week follow-up period⁴². This trend highlights that, despite hepcidin suppression by hypoxia and erythropoietic signaling, the inflammatory response predominates during the initial phase of infection, thereby limiting iron availability and promoting functional iron deficiency.

The reduction in iron availability for erythropoiesis is also exacerbated by pneumonia-causing bacteria, which employ diverse iron-acquisition strategies. A key mechanism involves the secretion of siderophores, high-affinity iron chelators capable of extracting iron from host carriers such as transferrin, lactoferrin, and hemoglobin, depriving the host of this essential element required for heme biosynthesis⁴³. This microbial iron competition may contribute to elevated FPP levels in pneumonia, reflecting the compounded effects of host-imposed iron sequestration and pathogen-driven iron scavenging.

In our study, FPP fluorescence was inversely correlated with serum iron, supporting that pneumonia-associated inflammation induces iron sequestration and restricts iron availability for erythropoiesis. However, serum iron levels alone lack prognostic value for mortality, as acute-phase responses, circadian variation, and nutritional status influence them. Similar findings were reported by Kjersti Oppen et al., who analyzed 267 hospitalized patients with CAP and found no significant associations between blood iron-related biomarkers and short-term adverse outcomes, including ICU admission or 30-day mortality³⁹. The potential superiority of FPP over traditional iron markers may arise from its ability to reflect iron availability in the bone marrow rather than merely circulating iron levels in peripheral blood, making it a more reliable indicator of iron-restricted erythropoiesis⁴⁴. Further supporting this, a significant positive correlation was observed between FPP levels and both the microR index and RDW, along with a significant negative correlation with MCHC, indicating a strong association between FPP and microcytosis/anisocytosis, which are hallmark features of iron-deficient erythropoiesis. These morphological abnormalities negatively impact erythrocyte rheology by reducing their deformability and impairing their ability to traverse the microvasculature efficiently, ultimately compromising oxygen delivery to tissue. In the context of pneumonia, this further exacerbates tissue hypoxia caused by impaired pulmonary gas exchange. As a result, the cardiovascular system compensates by increasing cardiac output and heart rate to maintain adequate perfusion. However, this adaptive response also raises myocardial oxygen demand, which may exacerbate cardiovascular strain, particularly in patients with preexisting heart disease or impaired coronary reserve. Our study observed a significant positive correlation between FPP and cardiac injury markers, specifically NT-proBNP and troponin, further reinforcing the link between iron-restricted erythropoiesis and increased myocardial stress.

The increased FPP levels in the blood can be directly attributed to stress erythropoiesis in the bone marrow, which prematurely releases immature erythrocytes reticulocytes into circulation as a compensatory response to pneumonia-induced hypoxia. Notably, one study confirmed that free protoporphyrin IX levels are significantly higher in reticulocytes, even when properly formed, compared to mature erythrocytes, further reinforcing the role of FPP as a marker of erythropoietic activity⁴⁵. This phenomenon is related to protoporphyrin IX being synthesized predominantly at the erythroblast stage. In contrast, the incorporation of iron into protoporphyrin IX to form heme continues in reticulocytes even after they enter the bloodstream.

Oxidative stress is an integral component of the inflammatory response in severe pneumonia. Activated leukocytes, particularly neutrophils and macrophages, generate large amounts of reactive oxygen species (ROS) as part of the immune defence against pathogens^46,47. While ROS play a crucial role in pathogen elimination, they can also cause collateral damage to host cells, including enzymes critical to essential metabolic pathways. Ferrochelatase, a key mitochondrial enzyme that inserts iron into protoporphyrin IX to form heme, is particularly susceptible to oxidative damage due to its iron-sulfur (Fe-S) clusters, which are highly sensitive to oxidation induced by inflammation and hypoxia⁴⁸. When ferrochelatase activity is compromised by oxidative stress, the incorporation of iron or zinc into protoporphyrin IX is disrupted, leading to the accumulation of free protoporphyrin IX in erythrocytes.

The accumulation of protoporphyrin IX in erythroblast mitochondria can result from disruptions in the finely tuned heme biosynthesis pathway⁴⁹. Under physiological conditions, aminolevulinate synthase 2 (ALAS2) catalyzes the condensation of glycine and succinyl-coenzyme A to form aminolevulinic acid, the first committed intermediate in the heme pathway⁵⁰. This multi-step cascade ultimately leads to heme synthesis, which exerts negative feedback on ALAS2 to prevent excessive protoporphyrin IX accumulation⁵¹. Heme formation is impeded when iron availability is compromised or when ferrochelatase activity is impaired. As a result, ALAS2 escapes heme-mediated feedback inhibition, allowing unchecked precursor flow and increased protoporphyrin IX production. Although an additional layer of translational control exists via an iron-responsive element (IRE) in the 5′ untranslated region (5′UTR) of ALAS2 mRNA, which suppresses ALAS2 translation under iron-deficient conditions, this mechanism does not fully compensate for the loss of feedback inhibition⁵². Consequently, protoporphyrin IX accumulates within erythroblast mitochondria, contributing to its elevated levels in the blood.

Zinc deficiency can also contribute to elevated FPP levels in pneumonia because the acute-phase response, driven by proinflammatory cytokines (IL-6, IL-1β, TNF-α), triggers metallothionein synthesis and sequesters zinc in the liver and immune cells⁵³. Additionally, macrophages and neutrophils, which exhibit an increased demand for zinc, actively take it up, thereby reducing its availability in the plasma and bone marrow, where heme synthesis occurs⁵⁴. Since ferrochelatase can incorporate zinc into protoporphyrin IX in iron-deficient states, zinc depletion further disrupts this alternative pathway, increasing free protoporphyrin IX accumulation. Simultaneously, nutritional disturbances, fever, and potential impairment of zinc absorption in the intestine may further exacerbate zinc deficiency, limiting the formation of ZnPP. Although higher dietary zinc intake (e.g., from meat) could theoretically influence ZnPP levels, inflammation-induced redistribution during pneumonia likely overrides such nutritional effects.

Some authors have proposed that the release of protoporphyrin IX may be mediated by interactions between pneumonia-causing pathogens and hemoglobin through structural proteins containing putative heme-binding domains^55,56,57. However, this hypothesis is primarily supported by computational modeling and lacks direct experimental validation.

Our study found a moderate correlation between FPP/F632 and the CCI, and a weaker association with age, suggesting that erythrocyte protoporphyrin content may partially reflect cumulative disease burden and age-related metabolic shifts. These alterations are likely linked to chronic inflammation, in which hepcidin-mediated iron sequestration impairs erythropoiesis, particularly in elderly or multimorbid patients.

A slight negative correlation between protoporphyrin fluorescence in erythrocytes and total cholesterol levels and a positive correlation with glucose levels has been observed. Although certain metabolic mechanisms may underlie these associations, this relationship remains insufficiently investigated. These correlations likely reflect parallel metabolic alterations induced by systemic inflammation. In hospitalized patients with pneumonia, low total cholesterol levels are often associated with acute-phase responses⁵⁸. The decline in cholesterol is driven by a metabolic shift toward the hepatic synthesis of acute-phase proteins, increased lipid uptake by activated immune cells, and enhanced cholesterol catabolism, an adaptive response to systemic inflammation. Conversely, elevated glucose levels in pneumonia patients are commonly attributed to the hypermetabolic and stress response associated with inflammation⁵⁹. Systemic release of proinflammatory cytokines, particularly TNF-α and IL-6, induces insulin resistance, impairing glucose uptake by peripheral tissues and enhancing hepatic gluconeogenesis⁶⁰. Additionally, activation of the hypothalamic-pituitary-adrenal (HPA) axis increases cortisol secretion, further promoting hyperglycemia⁶¹.

The 100-day follow-up was deliberately chosen to capture late complications and deaths that may stem from pneumonia. Extending observation beyond the usual 30-day window reveals longer-term risk in patients with substantial comorbidity, but it also blurs causal attribution: fatalities occurring two or three months after discharge are not always the direct result of pneumonia. In many cases, CAP precipitates acute decompensation of pre-existing conditions such as heart failure, chronic obstructive pulmonary disease, or malignancy, making it difficult to separate direct from indirect contributions to mortality. Prior studies show that sustained systemic inflammation and functional decline after CAP can raise death rates well beyond the acute phase^{62,63,64,65,66}. Despite this complexity, FPP/F632 remained an independent predictor of 100-day mortality after adjustment for age and comorbidity, suggesting that the fluorescence signal captures overall patient vulnerability rather than pneumonia alone. Future prospective work should incorporate formal adjudication of death certificates and hospitalization records, and include additional confounders to disentangle direct and indirect pathways linking CAP to late mortality.

The study cohort consisted of patients with moderately severe CAP who presented with a relatively uniform and apparently stable clinical picture at admission. In this setting, traditional bedside assessment alone offers limited granularity, particularly for long-term mortality stratification, underscoring the potential added value of fluorescence-based biomarkers. Prospective validation with complete clinical and laboratory data is planned to allow direct, head-to-head comparison between fluorescence-derived risk stratification and established clinical scoring systems such as CURB-65, PSI, and others.