Reduced global cerebral oxygen metabolic rate in sickle cell disease and chronic anemias

Abstract

Anemia is the most common blood disorder in the world. In patients with chronic anemia, such as sickle cell disease or major thalassemia, cerebral blood flow increases to compensate for decreased oxygen content. However, the effects of chronic anemia on oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO₂) are less well understood. In this study, we examined 47 sickle-cell anemia subjects (age 21.7±7.1, female 45%), 27 non-sickle anemic subjects (age 25.0±10.4, female 52%) and 44 healthy controls (age 26.4±10.6, female 71%) using MRI metrics of brain oxygenation and flow. Phase contrast MRI was used to measure resting cerebral blood flow, while T₂-relaxation-under-spin-tagging (TRUST) MRI with disease appropriate calibrations were used to measure OEF and CMRO₂. We observed that patients with sickle cell disease and other chronic anemias have decreased OEF and CMRO₂ (respectively 27.4±4.1% and 3.39±0.71 mL O₂/100g/min in sickle cell disease, 30.8±5.2% and 3.53±0.64 mL O₂/100g/min in other anemias) compared to controls (36.7±6.0% and 4.00±0.65 mL O₂/100g/min). Impaired CMRO₂ was proportional to the degree of anemia severity. We further demonstrate striking concordance of the present work with pooled historical data from patients having broad etiologies for their anemia. The reduced cerebral oxygen extraction and metabolism are consistent with emerging data demonstrating increased non-nutritive flow, or physiological shunting, in sickle cell disease patients.

Introduction:

Although the brain only accounts for 2% of the body mass, it accounts for 20% of the body’s resting energy expenditures.¹ Since the brain is unable to survive under anaerobic conditions, cerebral oxygen delivery is tightly regulated. Over 98% of the oxygen delivered to the brain is transported by hemoglobin.² Low hemoglobin level, or anemia, is common in the general population.³ Anemia is a hallmark of many genetic diseases worldwide, including sickle cell disease (SCD) and thalassemia.

Hemoglobin level is inversely correlated with cerebral blood flow (CBF) among healthy subjects.^4–6 To compensate for chronically decreased oxygen capacity, resting CBF rises to preserve global oxygen delivery.^7,8 Despite having adequate resting oxygen delivery, chronically anemic subjects demonstrate diffuse brain volume loss and silent strokes, suggesting chronic or acute-on-chronic supply-demand imbalance.^9–13 Early prior works in SCD T₂-relaxation-under-spin-tagging (TRUST)¹⁴ MRI with a calibration curve derived from bovine blood argued that brain oxygen extraction fraction (OEF) in SCD patients is increased proportional to the degree of anemia^15,16 to maintain normal cerebral metabolic rate of oxygen (CMRO₂).^17,18

Recent in vitro calibration work demonstrated unbiased and improved limits of agreement in OEF measurement using sickle-specific calibration compared to calibration derived from bovine blood.^19,20 Using these more appropriate calibration curves for human hemoglobins A and S, these works have demonstrated decreased OEF and CMRO₂ in SCD patients compared to healthy subjects.^21,22 Therefore, in light of these divergent and controversial findings, this study evaluated CMRO₂ using a recently published consensus calibration derived from sickle blood¹⁹ and compared venous oximetry, oxygen extraction and metabolic rate in a large cohort of control subjects, SCD and non-sickle anemic patients. We also systemically reviewed CMRO₂ in anemic patients performed using invasive, gold standard, MRI-independent assays (Positron Emission Tomography and Kety-Schmidt dilution technique²³).

Methods:

1. Study population:

All studies were performed at Children’s Hospital Los Angeles and approved by the Committee on Clinical Investigation (CCI#2011–0083). A total of 118 participants between 12 and 63 years of age were separated into three groups: healthy control subjects (CTL, N=44), subjects with sickle cell disease (SCD, N=47), and subjects with non-sickle chronic anemia (ACTL, N=27). Out of 118 subjects, 10 controls, 18 SCD and 7 ACTL subjects were between 12–18 years of age. Controls were drawn from families and friends of patients with SCD, so sickle cell trait was common, occurring in 23/44 control subjects. The ACTL group consisted of patients with thalassemia major (N=14), thalassemia intermedia (N=3), aplastic anemia (N=1), autoimmune hemolytic anemia (N=1), hereditary spherocytosis (N=3), hemoglobin H constant spring (N=4) and congenital dyserythropoietic anemia (N=1). The SCD group consisted of 39 subjects with SS hemoglobin, six subjects with SC hemoglobin, one with Sβ⁰ hemoglobin and one with Sβ⁺ hemoglobin. Chronic transfusion therapy was administered to 16/47 SCD patients and 16/27 ACTL patients every 3–4 weeks. Transfused SCD and ACTL patients were studied immediately prior to transfusion. Eleven non-transfused SCD subjects were undergoing hydroxyurea treatment at the time of the study. Exclusion criteria included: 1) pregnancy; 2) hypertension; 3) diabetes; 4) stroke or other known neurologic insult; 5) seizures; and 6) known developmental delay or learning disability. Group demographic and clinical variables are summarized in Table 1.

Table 1.

Subject demographics and hematologic markers. Bold lettering denotes statistically significant p-values (p < 0.05).

	CTL (N = 44)	ACTL (N = 27)	SCD (N = 47)	p-value (ACTL–CTL)	p-value (SCD–CTL)	p-value (SCD–ACTL)
Age (Years)	26.4±10.6	25.0±10.4	21.1±7.1	0.81	0.07	0.39
Sex	14M, 30F	13M, 14F	26M, 21F	0.37	0.06	0.82
Body Mass Index	25.7±7.0	23.1±2.5	22.3±4.4	0.11	<0.01	0.79
Transfused	0/44	16/27	16/47	<0.01	<0.01	0.02
Arterial Saturation (%)	99±1	99±1	98±2	0.23	<0.01	0.01
Systolic Blood Pressure (mmHg)	118±13	113±9	112±11	0.16	0.04	0.97
Diastolic Blood Pressure (mmHg)	68±10	62±7	60±7	<0.01	<0.01	0.55
Mean Blood Pressure (mmHg)	85±13	80±7	78±9	0.09	<0.01	0.83
Hemoglobin Electrophoresis	21AA, 23AS	23AA, 3AE, 1AS	39SS, 6SC, 1Sβ0, 1Sβ+
Hemoglobin (g/dL)	13.5±1.2	10.6±2.6	9.7±1.8	<0.01	<0.01	<0.01
Hematocrit (%)	40.0±3.2	32.5±6.2	27.6±4.7	<0.01	<0.01	0.07
White Blood Cell Count (x103)	5.7±1.8	6.9±2.3	9.7±4.3	0.26	<0.01	<0.01
Platelet Count (x103/µL)	251±56	268±118	321±128	0.79	<0.01	0.09
Reticulocytes (%)	1.3±0.5	2.6±2.8	9.2±5.0	0.30	<0.01	<0.01
HbS (%)	20±19	0	55±29	<0.01	<0.01	<0.01
Fetal Hemoglobin (%)	0.4±1.8	2.2±4.0	8.4±8.7	0.46	<0.01	<0.01
Cell-free Hemoglobin (mg/dL)	6.3±5.3	16.9±18.0	21.3±20.0	0.02	<0.01	0.49
Red Blood Cell Distribution Width	13.6±1.7	17.9±5.4	18.6±3.8	0.01	<0.01	0.76
Silent Cerebral Infarct Presence	9/44	8/27	20/47	0.63	0.04	0.25
CBF (mL/100g/min)	61.8±9.8	83.0±16.4	97.8±24.9	<0.01	<0.01	0.01
O2 Content (mL O2/mL blood)	18.2±1.5	14.4±3.4	13.0±2.4	<0.01	<0.01	0.05
O2 Delivery (mL/100g/min)	11.2±1.9	11.5±1.9	12.3±2.3	0.75	0.07	0.44
OEF (%)	36.1±5.8	30.9±5.6	27.5±4.1	<0.01	<0.01	0.03
CMRO2 (mL O2/100g/min)	4.01±0.63	3.53±0.65	3.39±0.71	0.01	<0.01	0.22

Imaging and blood samples were obtained on the same day for each subject. Blood gas analysis, complete blood count and quantitative hemoglobin electrophoresis were analyzed in our clinical laboratory.

2. MRI:

All imaging was performed on a Philips Achieva 3T MR system with an eight-channel, receive-only head coil. A 3D T1-weighted, T2-weighted fluid-attenuated inversion recovery (T2-FLAIR) 3D image and MR angiography image were acquired; details can be found in previous work.^24,25

Grey matter volume, white matter volume, cortical thickness and cortical surface area were calculated from T1 images based on previously-published methods.⁹ Silent cerebral infarcts equal or greater than 3 mm in diameter in two orthogonal planes^26,27 were documented on T₂-FLAIR images by the consensus of a neuroradiologist and neuroanatomist. Subjects with more than one silent cerebral infarction per decade of life were considered to have an abnormal burden of silent stroke.²⁸ 3D MRI angiography of the Circle of Willis and distal internal carotid artery was performed using standard techniques and assessed by a neuroradiologist for vasculopathy.

Whole-brain CBF was measured using phase contrast MRI;²⁹ details of phase contrast MRI acquisition has been explained in previous work.^8,30 Cerebral venous oxygenation Y_v was measured using TRUST MRI. The TRUST sequence used in this study has been explained in prior publications.^14,21,22 Briefly, a transverse, single-slice TRUST sequence with Carr-Purcell-Meiboom-Gill (CPMG) T₂ weighting was used to measured blood T₂ in the sagittal sinus. A sickle-specific calibration model was used to convert blood T₂ to venous saturation in SCD group;¹⁹ it reflected a consensus calibration derived from two independent studies.^18,21 A calibration model from human AA blood²² was used for control and non-sickle anemic patients. This calibration has been independently validated.¹⁸ Chronically transfused patients were characterized using a mixture model¹⁹ between the hemoglobin A and hemoglobin S calibration curves based upon their hemoglobin electrophoresis results at the time of the study. Although patients with sickle cell trait have up to 45% sickle hemoglobin, they have 0% sickle cells so their blood oximetry is accurately described by the hemoglobin A model.²² A summary of calibration models and their usage are shown in Supplemental Table S1.

3. Physiological parameters:

Several physiological parameters were derived using the following equations:

$$O_{\text{2}}\mathit{content}\text{=}\text{1.34}\text{×}\mathit{Hb}\text{×}{Y}_{\text{a}}\text{+}\text{0.003}\text{×}{\mathit{pO}}_{\text{2}}(mL{O}_{\text{2}}/mL\mathit{blood})$$ [1]

$$\mathit{OEF}\text{=}\frac{Y_a\text{–}{Y}_v}{Y_a}$$ [2]

$$O_{\text{2}}\mathit{delivery}\text{=}\mathit{CBF}\text{×}{O}_{\text{2}}\mathit{content}(mL/100g/min)$$ [3]

$${\mathit{CMRO}}_{\text{2}}\text{=}\mathit{CBF}\text{×}\mathit{OEF}\text{×}{O}_{\text{2}}\mathit{content}(mL{O}_{\text{2}}/100g/min)$$ [4]

where pO₂ is the partial pressure of oxygen estimated to be 100mmHg, Hb is the hemoglobin level, Y_v is the venous saturation measured by TRUST and Y_a is the arterial saturation measured by pulse oximetry.

4. Peripheral laboratory and venous blood gas measurements:

To probe for evidence of impaired oxygen extraction outside the brain, we performed blood-gas analysis and co-oximetry measurements of oxygen saturation from the brachial circulation. These measurements also have the advantage of being independent of MRI calibration and underlying disease state. A 22-gauge intravenous catheter was placed in the antecubital vein, and the tourniquet was released. After the intravenous line was secured (taking approximately 3–4 minutes), 15 mL of blood was drawn for a complete blood count, hemoglobin electrophoresis, and blood rheology. Following this, 0.5 ml was drawn slowly into a 1 ml blood gas syringe for analysis of venous blood gas (Alere Inc., EPOC Blood Analysis System, Waltham, MA) and oxygen saturation (Radiometer, OSM-3, Copenhagen, Denmark).

5. Reference data:

In addition to MRI data acquired in this study, we performed a literature review of studies that measured CMRO₂ using Kety-Schmidt²³ or Positron Emission Tomography (PET) in anemic patients and healthy subjects. Since we wanted to assess these reference datasets in comparison with our results, we limited our search to publications of various types of anemia that provided data of individual patients. Required parameters included hemoglobin, age and CMRO₂ values, or a complete set of measurements that could be used to calculate these parameters. This literature search was performed on PubMed, limited to papers published after 1900 and excluded studies that were related to animal work, infants, neonates and any other pathophysiology other than anemia.

6. Statistical analysis:

Statistical analysis was performed in JMP (SAS, Cary, NC). One-way ANOVA with Dunnett’s post hoc correction was used to examine the difference in parameters between SCD and control, and between ACTL and control; independent samples t-test was used to compare SCD and ACTL groups. Univariate and stepwise multivariate regressions were performed against hemoglobin, CBF, HbS, transfusion status, fetal hemoglobin, white blood cells, platelets, reticulocytes, cell-free hemoglobin and red blood cell distribution width and other imaging biomarkers including white matter volume, cortical thickness, cortical surface area and presence of silent cerebral infarcts; multivariate predictors were retained in the final model for p<0.05. Correction for age and sex were performed by removing the linear relationship with the measurement and then adding the measurement average back into the residuals to ensure final values were within physiologically meaningful range.

7. Data sharing statement:

For individual de-identified data, please contact the corresponding author.

Results:

1. Current study:

Table 1 summarizes the demographics for three groups. Since the control group trended slightly older and had more females compared to anemic subjects, subsequent analyses of OEF and CMRO₂ measures were corrected for age and sex. There were no significant differences in OEF and CMRO₂ between patients with hemoglobin SS, SC, Sβ⁺ and Sβ⁰ (p=0.92 for OEF, p=0.57 for CMRO₂) so all genotypes were pooled together. Control subjects with and without sickle cell trait had indistinguishable physiologic and laboratory values except for hemoglobin electrophoresis results, so data for hemoglobin AS and AA were pooled for all comparisons.

Anemic patients had lower hemoglobin, hematocrit and blood pressure compared to controls. Patients with SCD had higher white blood cells, platelets, reticulocytes, and fetal hemoglobin compared to ACTL subjects at the same hemoglobin level. Transfused SCD subjects had HbS percentages of 23±15%, whereas non-transfused patients had HbS percentages of 71±20%. Although no subject had a clinical history of stroke, T₂-FLAIR MRI showed silent cerebral infarct presence in nine control subjects (20%), 20 SCD subjects (43%, p=0.04) and eight ACTL subjects (30%, p=0.63). MRI angiography was completely normal in all subjects with no evidence of intracranial or extracranial vasculopathy.

Figure 1 compares oxygenation measures between three groups, and average oxygen parameters are summarized in Table 1. CBF was significantly higher in chronic anemias than normal controls (Figure 1A) but cerebral oxygen delivery was identical across groups (Figure 1B). Cerebral OEF was decreased in anemic subjects (SCD to a greater extent than ACTL) compared with control subjects (Figure 1C). CMRO₂ values were also decreased in SCD and ACTL groups compared with healthy controls (Figure 1D). OEF and CMRO₂ values were independent of transfusion status in SCD and ACTL subjects.

Figure 1.

Boxplot and linear correlations of oxygen supply and utilization values. Chronically anemic subjects demonstrated (A) increased CBF, (B) similar oxygen delivery but (C) lower OEF and (D) lower CMRO₂ compared to healthy controls. Linear correlation (E) between CMRO₂ and hemoglobin and (F) between cerebral OEF and brachial OEF in the cohort in this study. * denoted statistical significance p<0.05; NS denoted no significant difference.

2. Effects of anemia on cerebral oxygenation:

On univariate analysis, cerebral OEF was positively correlated with hemoglobin (r²=0.24, p<0.01) and negatively correlated with CBF (r²=0.26, p<0.01) and HbF (r²=0.11, p<0.01). Upon multivariate analysis, hemoglobin and HbF explained 24% and 5% of the variability respectively. Univariate and multivariate results are summarized in Supplemental Table S2.

Figure 1E demonstrate that CMRO₂ was moderately correlated with hemoglobin (r²=0.17, p<0.01). When the impact of hemoglobin was removed by linear regression, CMRO₂ was no longer significantly different between three groups (p=0.35), nor were any hematologic parameters retained as significant predictors. Neither OEF nor CMRO₂ were significantly correlated with imaging biomarkers of damage including white matter volume, cortical thickness, cortical surface area and presence of silent cerebral infarcts (Supplemental Table S2).

3. Peripheral oxygenation:

Brachial OEF was more variable than cerebral OEF, with an interquartile range of 18–61%, but was correlated with TRUST-based cerebral OEF (r²=0.17, p<0.01, Figure 1F). SCD and ACTL patients had brachial OEF of 29±22% and 37±23% respectively, significantly lower compared to control subjects (60±23%, p<0.01). Brachial OEF also demonstrated a positive correlation with hemoglobin (r²=0.14, p<0.01). After correcting for hemoglobin, brachial OEF was still significantly lower in transfused subjects but no longer different between the anemic and control groups (p=0.57, Supplemental Table S2).

4. Reference data:

Literature search yielded a total of 37 reports on brain oxygen metabolism of various pathophysiologies that included individual patient data, 28 of which reported both hemoglobin and CMRO₂. Of these publications, only eight studies focused on subjects with anemia and healthy controls. Details on these historical references can be found in Supplemental Table S4.

The eight studies included Herold et al.³¹ who studied SCD and control subjects using PET to demonstrate decreased CMRO₂ in SCD. Similarly, Frackowiak et al.³² also measured oxygen metabolism using PET in healthy volunteers. Using a nitrous oxide Kety-Schmidt technique,²³ Heyman et al.³³ studied sickle cell anemia, iron-deficiency anemia, aplastic anemia and anemia due to blood loss. With the same method, Scheinberg et al. studied oxygen utilization in patients with pernicious anemia³⁴ and healthy controls.³⁵ Stewart et al.³⁶ also studied oxygen circulation in pernicious anemia. Fazekas et al.,³⁷ Mangold et al.³⁸ and Kety et al.³⁹ published on cerebral hemodynamics on healthy controls under different observation conditions; only control datasets under baseline conditions were included to compare with CMRO₂ in anemia. Details on values extracted from the 8 references are shown in Supplemental Table S3.

The historical data are summarized in Figure 2. Figure 2A reveals a clear decrease in CMRO₂ with age (r²=0.20, p=0.01), making it necessary to perform age adjustment for all comparisons. Figure 2B demonstrates that age-adjusted CMRO₂ is directly proportional to hemoglobin level (r²=0.31), mirroring our present observations using MRI (Figure 1E). Figure 2C demonstrates that CMRO₂ was depressed in all anemia subjects. After controlling for hemoglobin level, no differences were observed among controls and all anemia subtypes. The same relationship with anemia severity remained even when the effects of varying arterial CO₂ tensions were accounted for (Supplemental Figure S1).

Figure 2.

Relationship between CMRO₂, age and anemia severity in historical references. (A) Linear correlation between CMRO₂ and age. (B) Linear correlation between age-adjusted CMRO₂ and hemoglobin. (C) Lower CMRO₂ in different anemia types compared to controls.

We further broadened our analysis of historical CMRO₂ data to 20 additional studies focused on specific diseases or conditions, where fluctuations in hemoglobin were a nuisance variable (Supplemental Table S4). An almost identical linear relationship between CMRO₂ and hemoglobin was observed (illustrated in Supplemental Figure S2). After controlling for variations in hemoglobin, the differences in CMRO₂ across studies were readily explainable by the primary disease process (such as hyperthyroidism, uremia, anesthesia, etc.).

In order to directly compare the results from our study with the historical references, we compared our MRI results to Figure 2A data in Figure 3. The scattergrams between CMRO₂ and hemoglobin were superimposed (Figure 3A). Figure 3B reveals that the MRI-based CMRO₂ measurements in both SCD and control groups corresponded quite well with results from Kety-Schmidt and PET. After controlling for hemoglobin, all differences among the subgroups were eliminated.

Figure 3.

Relationship between CMRO₂ and anemia severity when pooling the data from this current study (blue) with historical references (red). (A) Linear correlation between CMRO₂ and hemoglobin. (B) Lower CMRO₂ in different anemia types compared to controls.

Discussion:

In this manuscript, we examined 118 subjects and identified a reduction in OEF and CMRO₂ in patients with chronic anemia compared to controls. Decreased CMRO₂ has been previously reported in a subset of this cohort.^8,40 In this current work, we demonstrate that decreased CMRO₂ is correlated with anemia severity, independent of transfusion status and anemia subtype. We further corroborated our observations by comparing our results to reference data from eight independent studies in 146 healthy controls and anemic patients using gold-standard nitrous-oxide method and PET. The blunted cerebral OEF observed in anemic subjects was mirrored by cooximetry-assayed peripheral venous oxygen extraction, suggesting that decreased extraction was neither confined to the cerebral circulation nor an unanticipated artifact of the TRUST cerebral oximetry technique. Importantly, this work observed decreased OEF and CMRO₂ over a wide range of anemic etiologies in otherwise healthy anemic subjects. This decreased cerebral oxygen consumption despite preserved global oxygen delivery⁴¹ illustrates poor oxygen supply-demand matching and can potentially lead to cerebral hypoxia and ischemia.^42,43 Therefore, given the high incidence of neurovascular disease and stroke in anemic patients, mismatches in oxygen delivery and consumption need to be monitored and may provide meaningful clues into stroke etiology in chronic anemia physiology.

Multivariate regression analysis demonstrated univariate correlations between hematologic parameters and OEF and CMRO₂ – with hemoglobin as a consistent and strong predictor of both cerebral and brachial oxygenation values. After controlling for the effects of varying anemia severity, most hematologic variables were no longer significant predictors, demonstrating that anemia is the underlying and unifying biomarker of decreased oxygen extraction and metabolism in our cohort. The significant association between both fetal hemoglobin and transfusion with impaired OEF is consistent with a left shift of the hemoglobin dissociation curve at the same pO₂ operating point.^2,44 Such correlations suggested that even though induction of high HbF (through hydroxyurea use) and chronic transfusion are popular SCD treatments and offered protection against disease severity,⁴⁵ these treatments should be accompanied by frequent monitoring of cerebral oxygenation and stroke risk. Additionally, the lack of correlation between silent infarct presence and reduced CMRO₂ could potentially be explained by the regional characteristics of infarcts. The majority of brain metabolism occurs in the grey matter,^46,47 whereas silent infarcts tend to happen in the deep white matter regions.^24,48 Therefore, there is no a priori reason to expect correlation between white matter silent infarcts and global CMRO₂ values, except for their respective associations of anemia severity.⁴⁹

Classically, CMRO₂ has been thought to be preserved via modulations in blood flow and oxygen extraction. In humans, studies of hemodilution show that flow reserves respond initially and most robustly to ischemia.⁵⁰ As flow reserves become exhausted, extraction reserves are accessed and symptomatic impairment occurs, as seen in carotid stentosis.^51,52 In more extreme animal models of hemodilution, oxygen extraction and metabolic rate typically remain in the normal range until oxygen content is lowered below 40%.^53–55 In this current cross-sectional study, the oxygen content in our anemic subjects was 72±14% of the control cohort, much higher than hemoglobin levels associated with increased OEF in animal studies. More importantly, our subjects have experienced anemia for decades, allowing possible chronic compensation such as vascular remodeling^56,57 and alterations in oxygen diffusional paths⁵⁸ to affect oxygen unloading in the microvasculature. Though a few animal studies have observed increased OEF in severe subacute (2–3 weeks) anemia, none have systemically explored longer, more mild exposure to anemia.^59–61 Lastly, acute hemodilution is only a surrogate for chronic anemia and does not mirror the physiological complexity of sickle cell disease, thalassemia or any of the anemia syndromes we studied.

Even though preservation of global oxygen delivery via elevated blood flow in patients with chronic anemias is well established,^7,62 our data in combination with historical data compiled in this manuscript demonstrate concretely that CMRO₂ is diminished in chronically anemic populations. This observation is seemingly contradictory to recent reports of elevated OEF in SCD subjects suggested by Asymmetric Spin Echo (ASE) MRI techniques⁶³ and Near Infrared Spectrometry (NIRS).⁶⁴ However, these techniques measure spatial averages of oxygenated and deoxygenated hemoglobin concentration to estimate oxygen saturation within the tissue, independent of blood flow. As evidence of the difference between flow-weighted OEF and tissue saturation, a study using optical measurements of blood flow and oxygenation in mouse cerebral cortex demonstrated that flow-weighted oxygen extraction is lower than spatially-averaged oxygen saturation and that this difference increases under hyperemic conditions.⁶⁵ Laser Doppler and jugular venous catherization studies also demonstrated reduced arteriovenous difference during hyperemia, contrary to the increased tissue OEF observed in hyperemic SCD subjects by ASE.^66–68 In addition, ASE weighs grey matter and white matter oxygen consumption equally through spatial averaging, while the whole brain CMRO₂ measured by flow-weighted TRUST is dominated by grey matter oxygen consumption. As a result, neither ASE nor NIRS can be used to infer whole brain oxygen consumption, and OEF differences between ASE and TRUST are to be expected. Thus, flow-weighted and spatially-weighted OEF techniques complement one another and provide a window into brain capillary flow dynamics.

Our patient cohort also has physiological differences that might account for lower OEF values relative to some other published works. Firstly, we had no evidence of cerebral vasculopathy, so patients were able to adequately increase their CBF to maintain resting oxygen delivery,⁸ unlike carotid stenosis. This absence of clinical vasculopathy could also explain the lack of significant correlation between OEF and other clinical manifestation of disease, such as the presence of silent cerebral infarcts. Secondly, 1/3 of our patients were on chronic transfusion therapy. Transfused blood causes a left shift in the hemoglobin dissociation curve because of the 2,3-DPG depletion during blood storage,⁴⁴ leading to a lower OEF for any brain pO₂. Our non-transfused SS and Sβ₀ patients were taking hydroxyurea with good F response, which also promotes lower cerebral oxygen extraction. In fact, hemoglobin F concentration was retained as a significant predictor of OEF. Thirdly, 7/47 SCD patients had a milder genotype (SC or Sβ⁺). Older, sicker SCD patients in other studies have more circulating dense red blood cells, causing greater increases in their p50 and a larger right shift in the hemoglobin dissociation curve.⁶⁹ With this right shift, one would expect larger OEF for the same brain pO₂ operating point⁷⁰ compared to the younger, healthier cohort with normal resting oxygen saturation in this current study. Lastly, our study used TRUST oximetry calibration curves specifically derived from normal human subjects and patients with sickle cell disease, rather than relying on calibrations derived from cow blood.^19,20,22 In fact, the SCD calibration used in this study represents a consensus from two independent investigations characterizing the relationships between hematocrit, blood T₂, and oxygen saturation in SCD patients.¹⁹

Our cerebral oxygenation results were in agreement with several other recent publications as well as an all-inclusive historical sample of PET and Kety Schmidt oximetry data. Vaclavu et al. demonstrated decreased metabolic rate in SCD subjects relative to controls using TRUST oximetry and appropriate calibration curves.⁷¹ Using MRI susceptometry (which is robust to hemoglobin subtype),⁷² Croal et al. demonstrated that OEF decreased proportionally with anemia severity in SCD patients.⁷³ Lastly, our observed dependence of CMRO₂ mirrored decades of observations in a broad range of anemia syndromes. After corrections for age and hemoglobin level, CMRO₂ was identical across disease type, study, measurement technique, and era. The relationship between CMRO₂ and hemoglobin was maintained when the analysis was generalized to other medical conditions in which anemia was a confounding variable.

The decrease in CMRO₂ in our results could have several physiological explanations. Previously we have shown diffuse brain volume loss,^10,74 higher than normal rates of silent cerebral infarcts,^13,75,76 impaired resting state functional neural activity^77,78 and impaired neurocognitive function in chronically anemic subjects compared to healthy controls.^79–81 It is plausible to speculate that these neurovascular biomarkers could be associated with decreased CMRO₂, although we were not able to demonstrate such an association in this paper. In SCD, low hemoglobin and acute anemic events are the strongest predictors of new silent stroke.⁸² Additionally, stroke is most common in children (~6 years old) when CMRO₂ is also the highest.⁸² Therefore, understanding the mechanism and clinical significance of diminished OEF and CMRO₂ is not merely an academic enterprise.

Low CMRO₂ could also be explained by a protective downregulation of metabolic activity in anemic patients. Downregulation of metabolism could be induced by chronically impaired oxygen carrying capacity and long-term exposure to hypoxia. Hypoxic hypometabolism has been demonstrated in animal models to conserve oxygen and protect against hypoxic ischemic injuries.⁸³ Even though humans are believed not to exhibit hypoxic hypometabolism,⁸³ a previous study has demonstrated a reduction in metabolic heat and body temperature in response to acute hypoxia in humans.⁸⁴ Additionally, a study on native dwellers of high-altitude regions has shown that humans exposed to chronic hypoxia demonstrate a reduction in brain glucose metabolism especially in cerebral regions responsible for higher cortical functions,⁸⁵ thus indicating that protective hypometabolism could be a contributor to decreased CMRO₂ in our anemic subjects.

However, we believe that the reduction in OEF and CMRO₂ observed in anemic subjects represents a predictable, physiologic consequence of compensatory hyperemia.^7,8 Hyperemia reduces capillary transit time^30,86,87 and favors shorter mean cerebral capillary path lengths.⁶⁵ Because microvascular oxygen unloading requires sufficient residence time in the capillary network for efficient oxygen extraction, elevated CBF limits oxygen extraction. The resulting physiology is analogous to the disruption of microvascular oxygen exchange in conditions such as arteriovenous fistula. Even though our subjects do not have anatomical arteriovenous malformations, microvascular cerebral shunting has been suggested in SCD patients²¹ and is associated with lower OEF.¹⁷ Importantly, this physiology does not appear to be limited to the cerebral circulation. Our venous brachial oxygenation data, as well as prior reports of arterialization of peripheral blood flow in SCD⁸⁸ suggests a common underlying cerebral and peripheral vascular response to chronic anemia, strengthening confidence in our methods and conclusions.

Furthermore, mathematical models of capillary oxygen transport provide insights into the reduced OEF with hyperemia.⁸⁹ Increased CBF causes higher heterogeneity of blood flow and transit time in microvascular networks, leading to nonlinear oxygen-metabolism coupling, decreased OEF and lower oxygen consumption.^89,90 Whereas some microvascular beds have normal capillary transit, other beds can demonstrate a phenomenon similar to physiological shunting with vasodilated vessels, abnormally high flow and inefficient oxygen unloading (Supplemental Figure S3). These shunt-like beds contribute more flow to draining veins, leading to low overall OEF. Chronically, anemia and hyperemia damage the microvasculature potentially exacerbating physiological increases in capillary transit time heterogeneity and leading to a state of lower microvascular oxygen availability.^91–94

Limitations:

Our study was cross-sectional, only reflected steady state conditions, and was biased toward young adults who have preserved resting oxygen delivery. Microvascular disease and impaired vascular reactivity increase with age in the general population⁹⁵ and may be accelerated in hemoglobinopathy patients.⁹⁶ SCD patients at our institution have low stroke rates compared to the SCD populations in other studies.^17,97 Thus, lower OEF might not have been evident in an older cohort. On the other hand, since our study did not include children younger than 12 years of age whose CMRO₂ values are higher compared to young adults, our results could not be fairly compared with other work on pediatric SCD subjects.⁹⁸ Additionally, our oximetry measurements assumed saturation values acquired in the sagittal sinus was similar to whole-brain venous saturation. Although this is an accurate assumption in healthy controls,⁹⁹ SCD patients may preserve cortical perfusion at the expense of deeper structures.⁴¹ The study would also have been strengthened by studying CMRO₂ after hemoglobin manipulations, such as transfusions or phlebotomy, to build inference for causality between hemoglobin levels and CMRO₂.

Our historical reference data was drawn from only eight studies because there was a limited number of papers that included data for individual patients. We mitigated this shortcoming in Supplemental Figure S2 by expanding our selection criteria to include disorders in which CMRO₂ was likely to be abnormal and were able to replicate the relationship between Hb and CMRO₂. Since newer studies tended to only report group means instead of individual values, our references in Figure 2 had to be drawn from older studies (1930s-1980s). Since these references were from ~50 years ago, differences in OEF and CMRO₂ values between historical data and data in the current study could still be expected, even within control subjects. Even though these references were acquired with gold-standard methods, it would be useful to obtain more recent datasets from different acquisitions methods and test sites to further assess the relationship between OEF, CMRO₂ and anemia severity.

Conclusion:

In summary, using a hemoglobin specific T₂ oximetry calibration,¹⁹ we demonstrated that patients with SCD and other chronic anemias have lower OEF and CMRO₂ than control subjects, proportional to their anemia severity. CMRO₂ data agreed well with previously published study cohorts. The attenuated oxygen extraction at lower oxygen delivery in chronically anemic patients can best be explained by relative increases in non-nutritive flow, however this hypothesis awaits further experimental validation.

Data Availability

De-identified data available upon request.