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Lactate dehydrogenase to carboxyhemoglobin ratio as a biomarker of heme release to heme processing is associated with higher tricuspid regurgitant jet velocity and early death in sickle cell disease

Background Chronic hemolysis is a hallmark of sickle cell disease (SCD). Intravascular hemolysis in particular is associated with severe vasculopathic complications including pulmonary hypertension (PH) and early mortality. Free heme causes oxidative damage and recently was identified as erythrocyte-derived Danger Associated Molecular Pattern (e-DAMP), associated with endothelial activation and vaso-occlusion in SCD (Belcher et al., Blood. 2014; Ghosh et al., J. Clin. Invest. 2013). Intravascular hemolysis is associated with elevated levels of serum lactate dehydrogenase (LDH). Heme catabolism leads to endogenous carbon monoxide (CO) production by heme oxygenase-1 (HO1), and CO is eliminated in exhaled breath. CO is transported primarily as the conjugate carboxyhemoglobin (HbCO), and end-alveolar CO (EACO) is an accepted proxy marker for its concentration in blood. We evaluated several lab values and ratios that might reflect the relative contribution of intravascular heme release and overall heme processing.
 Methods We investigated the relationship between EACO, HbCO (NCT01547793, cohort A) and other biomarkers of hemolysis in adults with SCD at steady state as part of the clinical cohort at the National Institutes of Health Clinical Center, Bethesda, Maryland, USA (NCT00011648, cohort B). Of the patients included in the cohort B, all routine samples with results on HbCO were included in the analyses. In a subgroup of the cohort B with data available on HbCO, echocardiography and/or mortality, we evaluated the correlation between LDH/HbCO ratio and echocardiographic markers of PH and all-cause mortality (cohort C).
 Combining all recognized available markers for hemolysis (total bilirubin, AST, absolute reticulocyte count, hemoglobin, median LDH, median HbCO and LDH/HbCO ratio) in a multivariate Cox proportional hazards model for survival led to selection of a predictive model encompassing three biomarkers: LD/HbCO ratio, AST and hemoglobin. Of these three markers, the LD/HbCO ratio was the most predictive factor. We also conducted univariate correlations with clinical outcome indicators.
 Main findings Erythropoietic and hemolytic laboratory parameters of the cohorts are provided in Table 1. HbCO concentrations and EACO were strongly correlated (Pearson's correlation r=0.66, p<0.01). In both cohort A and cohort B, HbCO and EACO were not correlated to LDH. However, EACO and HbCO did correlate with absolute reticulocyte counts (respectively r=0.46, p<0.01 and r=0.58, p<0.01).
 The patients of cohort C were divided into low (peak TRV <2.5m/s, N=34), intermediate (peak TRV 2.5-3m/s, N=38) and high risk (peak TRV >=3.0m/s, N=13) categories, based upon prior cut-points determined by risk of development of PH and early mortality (Mehari A. et al. JAMA. 2012) (Figure 1, panel A). LDH/HbCO ratios were positively correlated with TRV (r=0.38, p<0.01), and were significantly higher in patients with TRV >=3.0m/s (Mann-Whitey U test; p=0.02). In contrast, LDH values alone were not discriminative. All patients (25/25) with a LDH/HbCO ratio <1,200 had a TRV <3.0m/s; 94% (15/16) of the patients with catheterization-proven PH had a LDH/HbCO ratio >1,200. In the intermediate risk subgroup, PH was only diagnosed in individuals with LDH/HbCO ratios exceeding 1,200.
 Median follow-up was 12.1 years (IQR 10.3; 16.3), 25% (23/91) of the patients died during follow-up. Five-year, 10-year and 15-year overall survival in the group with LDH/HbCO ratio >1,200 were respectively 92.1%, 76.0% and 69.1%, whereas 5-year, 10-year and 15-year overall survival in the group with LDH/HbCO ratio <1,200 were respectively 100%, 92.9% and 88.0% (Figure 1, panel B). LDH/HbCO ratios were associated with all-cause mortality in a Cox proportional hazards model (p<0.01) and remained significantly associated with all-cause mortality when adjusted for age, C-reactive protein and ferritin (p=0.02). LDH alone was not associated with all-cause mortality in the unadjusted analysis.
 Main conclusions A ratio of two readily available clinical laboratory markers, LDH and HbCO, is promising as a potential biomarker in SCD. Increased LDH/HbCO ratios are strongly associated with elevated TRV and all-cause mortality, and thereby might improve the individual risk prediction in SCD patients. These markers deserve additional validation in future prospective trials.
 
 
 
 van Wijk: Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Minniti:Doris Duke Foundation: Research Funding. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding. van Beers:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding; Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.


Lactate dehydrogenase to carboxyhemoglobin ratio as a biomarker of heme release to heme processing is associated with higher tricuspid regurgitant jet velocity and early death in sickle cell disease To the Editor: Chronic hemolysis is a hallmark of sickle cell disease (SCD). Prior estimates of intravascular hemolysis as a proportion of the total hemolytic rate ranged from less than 10% to more than 30%. Chronic intravascular hemolysis is associated with severe vasculopathic complications including pulmonary hypertension and early mortality in SCD. 1 Intravascular hemolysis leads to release of cellular free hemoglobin and heme. Normally, these are scavenged by respectively haptoglobin and hemopexin. However, in SCD patients the availability of scavenging proteins in plasma is typically decreased because of rapid consumption by the prodigious amount of intravascular hemolysis.
Both cellular free hemoglobin and heme, devoid of anti-oxidant buffering mechanisms normally present in red cells, 2 are recognized as erythroid damage-associated molecular pattern molecules (e-DAMPs) and contribute to development of chronic vasculopathy, platelet activation and pulmonary hypertension in SCD. Intravascular heme release is associated with elevated levels of serum lactate dehydrogenase (LDH). 1 All heme metabolized either via extravascular hemolysis or cleared from the plasma via scavenger proteins, will lead to endogenous carbon monoxide (CO) production by hemeoxygenase 1 (HO-1). In contrast to heme, CO has many protective effects including inhibition of polymerization of sickle hemoglobin, increased red blood cell hydration, anti-oxidative and antiinflammatory effects. 3 Theoretically, the balance of detrimental intravascular hemolysis to beneficial CO production (total hemolysis) could be a prognostic biomarker in SCD. Because the vast majority of endogenous CO is cleared by exhalation, end-alveolar CO (EACO) in morning first breath is an accepted biomarker of endogenously produced CO, and thereby an accepted proxy marker of overall hemolysis. Before exhalation, CO is transported primarily as the conjugate carboxyhemoglobin (HbCO).
Here we explore whether plasma HbCO can be a convenient surrogate marker of endogenous CO production. In addition, we hypothesize that the ratio of LDH to CO may give an estimate of the relative contribution of intravascular heme release to total heme processing and relates to clinical endpoints, and therefore could be a new readily available biomarker of interest in SCD.
We investigated the relationship between EACO to HbCO plasma levels (n = 37; cohort [A]). After establishing a good correlation, we evaluated the relation of the ratio of LDH/HbCO to echocardiography parameters and mortality rates in adults with SCD at the NIH, A thorough description of study population, trial conduct and statistical analysis is provided in supplemental section 1.
An overview of the cohorts' demographics, hematological and hemolytic parameters is provided in Table S1.
We confirmed the expected correlation among surrogate biomarkers of intravascular heme release (LDH, AST).  (Table S2). Additionally, we explored variability in CO production in individual patients ( Figure S2). The observations suggest that every patient has a finite HbCO range with minor variation over time and not significantly associated with changes in LDH in this limited dataset. Thus, the serum level of LDH (known to reflect intravascular hemolysis and other tissue injury, but not extravascular hemolysis) appears to be heterogeneous among SCD patients and does not directly represent total hemolysis as represented by HbCO. This interpretation led us to formulate that the fraction of total hemolysis that is intravascular might more directly correlate with vascular health outcomes.
Next, we investigated whether an increased fraction of intravascular hemolysis, that is an increased ratio of LDH to HbCO, was All-cause mortality predictability by LDH/HbCO ratio and LDH was analyzed in a Cox proportional hazard model (Table S3). The LDH/HbCO ratio was significantly associated with mortality in the unadjusted analysis (p < 0.01). Age, CRP and ferritin were previously reported to be related to early-mortality in SCD. In our cohort age (p = 0.03), CRP (p = 0.05) and ferritin (p < 0.01) were associated with mortality, as was TRV (p < 0.01). In the multivariate-adjusted analysis including age, CRP and ferritin, LDH/HbCO ratios remained significantly associated with all-cause mortality (p = 0.02). There was a clear trend toward association between LDH and all-cause mortality in the unadjusted analysis (p = 0.10), however, it did not reach statistical significance in our cohort. Kaplan-Meier survival probabilities for patients stratified by LDH/HbCO ratios greater than and less than 1200 are shown in Figure 1.
In summary, the LDH/HbCO ratio predicts survival probability independent of age, ferritin and CRP.
The products of intravascular red cell lysis have shown to be of major importance in the pathophysiology of vasculopathy in SCD.
Infusion of high dose hemoglobin or heme in SCD mice induces lethal vaso-occlusive events. 4 In contrast, low-dose heme and subsequent induction of HO-1 and production of the gasotransmitter CO is protective against vaso-occlusion in SCD mice. 4 Therefore, it is biologically plausible that the LDH/HbCO ratio, representing an increase in the amount of CO, as product of heme processing, relative to the as an indication of total hemolysis. However, the amount of excreted heme is limited compared to total red cell turnover. Therefore, we consider that HbCO is generally reflective of the total amount of CO production due to adaptive heme catabolism.
Serum LDH has been the most widely used marker of intravascular hemolysis associated with catheterization-proven pulmonary hypertension or elevated TRV in SCD cohorts. Elevated TRV in SCD was significantly associated with mortality in a recent meta-analysis, but an inconsistent association was observed between LDH and mortality. 5 Notably, the current cohort on whom we have CO, LDH and mortality data is a subset with less statistical power than some of the prior datasets. As such, this present study may be underpowered to replicate prior LDH-mortality associations. Although all patients diagnosed with SCD were eligible for enrollment in these trials our cohort was enriched for SCD patients at risk for vasculopathic complications and/or early death. In this relatively small cohort, we show that the ratio of LDH to HbCO is superior to predict either elevated TRV or mortality as compared to LDH alone. These results suggest that the LDH/HbCO ratio may enhance the utility of LDH to identify patients at risk for vasculopathic complications and early death in SCD.
One limitation of our study is the presentation of HbCO values in relation to other hemolytic parameters in two cohorts that differ in regard to patient selection and size. The EACO cohort (A) was prospective and smaller; and, the HbCO cohort (B) was larger but retrospective and, therefore, may be biased toward a more acutely or chronically affected subpopulation for whom HbCO data were available. Altogether, HbCO results must be considered exploratory and in

DATA AVAILABILITY STATEMENT
The aggregated data and analyses that support the findings of this study are available upon request from the corresponding author.
Other data related to the patients are curated at the NIH, NHLBI,