Clinical implication of changes in serum cations and anions on clinical severity in sickle cell disease: A case–control study in a tertiary center

INTRODUCTION

Sickle cell disease (SCD), an autosomal recessive hemoglobinopathy, is characterized by sickle-shaped red blood cells (RBCs). The most common complications in these patients are chronic hemolysis, microvascular occlusion, and tissue damage, and all stem from the tendency of sickle cell hemoglobin (HbS) to polymerize when deoxygenated.^[1,2] Repeated bouts of hypoxia and dehydration make red cells stiff and sticky. An increase in stiffness leads to splenic entrapment and extravascular hemolysis leading to microvascular occlusion. Such repeated events cause ischemia-reperfusion insult to organs leading to organ damage.^[1,3] Recent articles reported that such abnormality could impair the potassium–chloride (K⁺-Cl⁻) cotransport mechanism and the calcium (Ca²⁺)activated Gardos channel in RBC membranes responsible for the intraerythrocytic shift of K⁺.^[4] In deoxygenated state, the RBC membranes become permeable to Ca²⁺ and permeable cation channels are activated, and the Ca²⁺ current flows into the cytosol. Rapid Ca²⁺ influx of ions into the cell causes abnormal ion channel activation, leading to the efflux of K⁺ ions and water from the RBCs.^[4-7] Thus, sickling raises intracellular Ca²⁺ and loss of K⁺ and water from red cells, thereby increasing K⁺ levels in the blood. K⁺ is the principal cation for heart and muscle function; its rise will lead to abnormal heart function and muscle weakness. An altered sodium–potassium ATPase (Na⁺-K⁺ ATPase) function, often seen in sickle cell patients, also creates serum electrolyte imbalance.^[3] A decrease in serum sodium (Na⁺) will impair nerve impulse conduction.^[3] Na⁺, one of the major cations of the extracellular space, is the crucial regulator of water homeostasis. Besides, it is critical in the muscular system and nerve conduction. Along with Na⁺, chloride (Cl⁻) also helps maintain body fluid. Alterations of these fluid ions might result in altered consequences.^[3] Intracellular dehydration in these patients increases mean corpuscular hemoglobin concentration (MCHC), which increases the probability of aggregation and polymerization during any given period of deoxygenation. SCD patients suffer from frequent episodes of hemolysis, managed mainly by repeated blood transfusions leading to electrolyte imbalance, most often hyperkalemia. Changes in plasma osmolality, either due to fluid overload during intravenous therapy or kidney dysfunction, leads to RBC dehydration, intracellular HbS concentration, and polymerization in the deoxygenated state, further promoting sickling.^[1,3,8] The extracellular tonicity alters the RBCs’ biomechanical properties; thus, managing these patients with suitable intravenous fluids of appropriate volume and flow rate would most likely improve the rheological properties and reduce the frequency of crisis events.^[9] Presently, Hb and fetal Hb (HbF) is routinely measured to assess the clinical course.^[10] HbF is routinely measured in highperformance liquid chromatography (HPLC) technique which is not only costly but also not feasible in all health care centers so that patients could be monitored. Therefore, monitoring the clinical course using markers that could be easily measured at all health care facilities would be more convincing. The study aimed to understand the ongoing changes in serum cations and anions and their impact on clinical severity in SCD patients. It is hypothesized that regular monitoring of serum ions will reduce mortality and morbidity and increase sickle cell patients’ life expectancy.

MATERIALS AND METHODS

The case–control study was conducted on 53 adult participants attending the sickle cell clinic of the institute. The study participants comprised 26 cases and 27 apparently healthy control individuals. The cases included all homozygous SCD (HbSS) diagnosed individuals after HPLC confirmation in the Variant-II Hemoglobin Testing System (BioRad). The subjects were further grouped as crisis state (n = 18) and steady state cases (n = 8) to understand the differences in serum parameters. Individuals presenting with either of the symptoms of crisis, as depicted in Table 1, were grouped under crisis state, and those who attended the sickle cell clinic for follow-up and had no such symptoms were grouped under steady state. Cases with a history of blood transfusion in the last 2 weeks were excluded to avoid the confounding results of biochemical changes associated with transfusion. The clinical details were recorded as per the validated questionnaire used in the clinic. As per the presenting symptoms, each participant grouped under cases was assigned a score as per the severity scoring system from Adegoke and Kuti.^[11,12] The scores were added, and a disease severity score was calculated for each. Blood samples were collected per the aseptic protocol in a plain vacutainer with a clot activator (red cap). Serum was analyzed for electrolytes (sodium, potassium, and chloride) by ion-selective electrode modules, calcium by Arsenazo III method, phosphorus by modified Daly and Ertingshausen method, magnesium by xylidyl blue method, total protein by modified Weichselbaum method, and albumin by bromocresol green method processed in AU 5811 fully automated autoanalyzer from Beckman Coulter. Serum osmolality was measured using the principle of freezing point depression in Advanced Osmometer (3250) (Advanced Instruments, Massachusetts, United States). Calculations for ratios and conversion to mmol/L were performed as given in Table 2.^[13-15]

The mutual relationships of the serum variables with each other and the impact on clinical severity, various ratios were considered during analysis like sodium–potassium ratio, sodium–chloride ratio, calcium–phosphorus ratio (CPR), calcium–magnesium ratio (CMR), cation–anion ratio (CAR), and sodium–albumin ratio (SAR). Since serum osmolality is a net effect of all cations and anions, including albumin in serum, ratios of osmolality with these serum variables were calculated, such as osmolality–sodium ratio (OSR), osmolality– albumin ratio (OABR), osmolality–cation ratio, and osmolality–anion ratio (OAR).

Statistical analysis

IBMSPSS version 26 was used for the statistical analysis. The quantitative variables were checked for normality, and the study groups’ values were compared by independent samples’ t-test [Tables 3 and 4]. The influence of the serum variables on disease severity score in cases, as depicted in Table 5, was analyzed by linear regression analysis. The serum variables in cases were analyzed by the receiver operating characteristics (ROC) curve and area under the curve (AUC) for the occurrence of the crisis event. From the ROC analysis, the cutoff values with the highest sensitivity for a case to present with a crisis state were determined and tabulated in Table 6 Figures 2 and 3. The frequency percentages of cases with normal and abnormal serum values were calculated and delineated in Figure 1. The distribution pattern between the crisis and steady states of the cases were compared using chi-square test and the odds ratio (OR) with a 95% confidence interval (95% CI).

Table 3: Comparison of quantitative variables between the study groups (N = 53)

Parameters (units)	Cases N = 26	Control N = 27	p-Value
	Mean (SD)
Age (y)	22.19 (6.23)	22.85 (2.58)	0.25
Sodium	135.65 (4.17)	140.56 (2.03)	0.005a
Potassium	4.31 (0.6)	4.24 (0.42)	0.101
Chloride	99.88 (5.9)	103.44 (2.06)	0.001a
Sodium–potassium ratio (SPR)	32.02 (4.3)	33.45 (3.2)	0.17
Sodium–chloride ratio (SCR)	1.36 (0.05)	1.36 (0.02)	0.013a
Osmolality	285.19 (9.15)	292.04 (6.09)	0.068
Calcium	2.28 (0.2)	2.45 (0.13)	0.027a
Phosphorus	1.39 (0.32)	1.2 (0.2)	0.08
Magnesium	0.76 (0.12)	0.86 (0.07)	0.004a
Calcium–phosphorus ratio (CPR)	1.72 (0.47)	2.12 (0.5)	0.55
Calcium–magnesium ratio (CMR)	3.07 (0.5)	2.85 (0.28)	0.007a
Total protein	7.45 (1.01)	7.89 (0.47)	< 0.001a
Albumin	0.59 (0.11)	0.69 (0.05)	0.001a
Cation–anion ratio (CAR)	1.44 (0.06)	1.43 (0.02)	0.013a
Cations	143.01 (4.2)	148.1 (2.1)	0.011a
Anions	101.87 (5.9)	105.34 (2.02)	0.001a
Sodium–albumin ratio (SAR)	239.59 (46.6)	204.3 (12.6)	0.001a
Osmolality–sodium ratio (OSR)	2.1 (0.06)	2.08 (0.05)	0.21
Osmolality–albumin ratio (OABR)	504.72 (104.1)	424.51 (27.3)	< 0.001a
Osmolality–cations ratio (OCR)	1.99 (0.06)	1.97 (0.04)	0.115
Osmolality–anions ratio (OAR)	2.85 (0.15)	2.81 (0.07)	0.005a

Abbreviation: SD, standard deviation.

^aSignificant at p value<0.05.

Table 4: Comparison of quantitative variables between the cases (N = 26)

Parameters	Crisis N = 18	Steady state N = 8	p-Value
	Mean (SD)
Age (y)	23.33 (7.2)	19.63 (0.7)	0.012a
Sodium	135 (4.8)	137.13 (1.6)	0.01a
Potassium	4.19 (0.6)	4.58 (0.7)	0.302
Chloride	99.8 (7.1)	100.13 (1.8)	0.008a
Sodium–potassium ratio (SPR)	32.7 (4.2)	30.48 (4.4)	0.72
Sodium–chloride ratio (SCR)	1.36 (0.06)	1.37 (0.02)	0.07
Osmolality	285.50 (10.7)	284.5 (4.5)	0.067
Calcium	2.21 (0.19)	2.45 (0.06)	0.011a
Phosphorus	1.46 (0.36)	1.27 (0.13)	0.002a
Magnesium	0.72 (0.12)	0.86 (0.07)	0.104
Calcium–phosphorus ratio (CPR)	1.63 (0.5)	1.94 (0.2)	0.049a
Calcium–magnesium ratio (CMR)	3.16 (0.5)	2.87 (0.3)	0.036a
Total protein	7.13 (0.99)	8.18 (0.58)	0.13
Albumin	0.535 (0.08)	0.698 (0.04)	0.14
Cation–anion ratio (CAR)	1.44 (0.07)	1.45 (0.02)	0.041a
Cations	142.1 (4.7)	145.01 (1.6)	0.023a
Anions	101.77 (7.09)	102.09 (1.8)	0.008a
Sodium–albumin ratio (SAR)	258.49 (43.7)	197.05 (11.3)	0.04a
Osmolality–sodium ratio (OSR)	2.12 (0.07)	2.08 (0.05)	0.66
Osmolality–albumin ratio (OABR)	547.3 (97)	408.97 (26.8)	0.036a
Osmolality–cations ratio (OCR)	2.01 (0.06)	1.96 (0.05)	0.57
Osmolality–anions ratio (OAR)	2.86 (0.17)	2.82 (0.09)	0.097

Abbreviation: SD, standard deviation.

^aSignificant at p value<0.05.

Table 5: Influence of the serum parameters on disease severity score in the cases (N = 26)

Parameters	Disease severity score
	Pearson’s correlation (r)	p-Value
Sodium	–0.089	0.332
Chloride	–0.018	0.47
Sodium–potassium ratio (SPR)	0.069	0.37
Sodium–chloride ratio (SCR)	–0.054	0.39
Osmolality	–0.057	0.39
Calcium	–0.367	0.033a
Phosphorus	–0.036	0.43
Magnesium	–0.285	0.079
Calcium–phosphorus ratio (CPR)	–0.025	0.45
Calcium–magnesium ratio (CMR)	0.094	0.32
Albumin	–0.338	0.046a
Cation–anion ratio (CAR)	–0.066	0.37
Cations	–0.129	0.265
Anions	–0.026	0.45
Sodium–albumin ratio (SAR)	0.328	0.05a
Osmolality–sodium ratio (OSR)	0.025	0.45
Osmolality–albumin ratio (OABR)	0.31	0.062
Osmolality–cations ratio (OCR)	0.055	0.39
Osmolality–anions ratio (OAR)	–0.029	0.44

Note: r Pearson’s correlation coefficient.

^aSignificant at p value<0.5.

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Figure 1:

Frequency percentage distribution of normal and abnormal serum parameters in the cases (N 26). ^mSignificant at p < 0.05; Na⁺, serum sodium; K⁺, serum potassium; Cl^-, serum chloride; Cal, serum calcium; Phos, serum phosphorus; Mg, serum magnesium; Osmol, serum osmolality; T. protein, serum total protein; Alb, serum albumin.

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Figure 2:

Receiver operating characteristics (ROC) curve for elevated levels of the serum parameters that influence the crisis state to occur. CMR, calcium–magnesium ratio; OABR, osmolality– albumin ratio; OSR, osmolality–sodium ratio; SAR, sodium– albumin ratio; SPR, sodium–potassium ratio.

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Figure 3:

Receiver operating characteristics (ROC) curve for reduced values of the serum parameters that influence the crisis state to occur. Na, serum sodium; Cal, serum calcium; Mg, serum magnesium; Alb, serum albumin; CAR, cation–anion ratio.

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RESULTS

The study subjects comprised of 53 adult participants that included 26 cases of HbSS and 27 apparently healthy individuals as the control group. The mean (standard deviation), as depicted in Table 3, serum sodium (135.65 [4.17], p = 0.005), chloride (99.88 [5.9], p = 0.001), calcium (2.28 [0.2], p = 0.027), magnesium (0.76 [0.12], p = 0.004), total protein (7.46 [1.01], p < 0.001), and albumin (0.59 [0.11], p = 0.001) mmol/L, were lower in cases than the contemporary control group. In cases, serum osmolality was low (285.19 [9.15] mOsm/kg, p = 0.068). In addition, the overall cations (143.01 [4.2], p = 0.011) and anions (101.87 [5.9], p = 0.001) mmol/L, were also significantly decreased in cases, and accordingly, the ratios such as CMR, CAR, SAR, OABR, and OAR were elevated in cases.

The serum parameters were compared between the two groups of the cases, crisis state and steady state groups, as shown in Table 4. A significant reduction in serum sodium (135 [4.8] mmol/L, p = 0.01), chloride (99.8 [7.1] mmol/L, p = 0.008), calcium (2.21 [0.19] mmol/L, p = 0.011), CPR (1.63 [0.5], p = 0.049), cations (142.1 [4.7] mmol/L, p = 0.023), anions (101.77 [7.09] mmol/L, p = 0.008), and CAR (1.44 [0.07], p = 0.041) was observed in cases compared with crisis state. On the contrary, raised serum phosphorus (1.46 [0.36] mmol/L, p = 0.002), CMR (3.16 [0.5], p = 0.036), SAR (258.49 [43.7], p = 0.04), and OAR (547.3 [97], p = 0.036) were evidenced in crisis state individuals.

These parameters were correlated for disease severity score in cases as delineated in Table 5. The findings suggested serum calcium (r =–0.367, p = 0.033) and albumin (r =–0.338, p = 0.046) correlated inversely, whereas SAR (r = 0.328, p = 0.05) depicted a positive influence on disease severity score.

The AUC deciphered through ROC analysis for the occurrence of crisis events in cases are revealed in Table 6 and Figures 2 and 3. A gross AUC was observed for raised levels of SAR (AUC = 0.986, p < 0.001) and OABR (AUC = 0.986, p < 0.001; not shown). Similarly, the AUC was 0.868 for low serum levels of calcium (p = 0.003) and 0.986 for low serum albumin levels (p < 0.001).

The cutoff values with 94.4% sensitivity for serum SAR and OABR were 208.45 and 433.79. The sensitivity for crisis events was 83.3 and 88.9%, respectively, for serum cutoff values of calcium and albumin at 2.41 and 0.63 mmol/L in cases.

The frequency percentage distribution of the normal and abnormal serum parameters in the cases is deciphered in Figure 1. Serum osmolality was found normal in all the steady state cases. However, in the crisis state group, low osmolality was noted in 11.1% and high osmolality in 16.7% of cases (chi-square = 2.751, p = 0.253). Abnormal serum osmolality would increase the probability of crisis state by nearly two times more than those with normal serum osmolality. The figure reflects that 50% of the cases presenting with a crisis state had low serum sodium, whereas none in the steady state had abnormal serum sodium values (chisquare = 4.89, p = 0.013). The OR was 11.59 (95% CI = 1.157– 389.7) for crisis events with low sodium than those within the normal reference range.

Similarly, low serum calcium and magnesium were noted in 38.9% (chi-square = 3.1, p = 0.039) and 44.4% (chi-square = 3.95, p = 0.023) of the crisis state cases, whereas none reported such lower values in steady state. The patients with low calcium were 7.52 times more prone to developing crisis episodes than those with normal serum values. On the contrary, raised serum phosphorus levels were recorded in 50% of the crisis state individuals compared with only 12.5% of steady state individuals (chi-square = 3.71, p = 0.03) with OR of 7.29 (95% CI = 0.87–197).

Of all the crisis state cases, 44.4% depicted low total protein and 83.3% represented low serum albumin (chisquare = 14.26, p < 0.001). None of the steady state cases had either low total protein or albumin. The chances for crisis were more than 50 times higher in individuals with low albumin than those with serum albumin within normal reference range (95% CI = 4.59–1864).

DISCUSSION

The case–control study included 27 apparently healthy adults and 26 HbSS SCD cases. The findings revealed a significant lowering of cations (p = 0.011) and anions (p = 0.001) in the cases than the control group [Table 3]. The serum osmolality was low in cases (p = 0.068), along with the significant extracellular ions contributing to serum osmolality, such as sodium and chloride. Antwi-Boasiako et al reported lower serum sodium and higher serum potassium in HbSS patients than in the control group (p < 0.001).^[3] Similarly, other studies also reflected reduced serum sodium and raised serum potassium in SCD cases than the healthy control group.^[16,17] Deoxygenation of the sickle cell RBCs is prone to altered cation permeability primarily attributed to red cell membrane Na⁺-K⁺ ATPase activity activation. Because of intracellular dehydration, the MCHC in sickle cell RBCs seems to be raised. The present study also observed a significant influence of serum osmolality with MCHC values (B = –0.110, p = 0.023; data not shown). The relative increase in HbS in these cells consequent to electrolyte imbalance results in rapid polymerization of the HbS. These events eventually result in vaso-occlusion in the microcirculation leading to crisis events and painful episodes. Brown et al study in a mouse model suggested that activated K⁺-Cl^- cotransport worsens the disease pathogenesis and tissue damage.^[5] Other articles also proposed that such abnormality could result in end-organ damage with increased morbidity like kidney dysfunction, cerebrovascular stroke, and mortality.^[4,5,18] The results of the present study, low serum sodium and chloride, agreed with the above findings [Table 4] and propose that dehydration prevention would help reduce the crisis episodes. Besides serum electrolytes, serum calcium and magnesium were also grossly reduced in the cases of their healthy contemporary group [Table 3]. In addition, serum calcium depicted a significant negative correlation with disease severity [Table 5]. The explanation is that intracellular dehydration results from the activation of Gardos channels resulting in Ca²⁺ influx and loss of K⁺ and water. The net result is raised intracellular Ca²⁺ that triggers the downstream signals in the red cells, such as osmotic fragility, cytoskeleton instability, and membrane asymmetry for phospholipids distribution.^[19,20] Magnesium, on the other hand, acts as a vasodilator and modulates the voltage-gated calcium channels. The resultant effect of hypocalcemia and hypomagnesemia is red cell dehydration and eryptosis (RBCs with features of senescence) followed by hemolysis and microvascular occlusions.^[20] Consequent to hemolysis, serum phosphorus also tends to rise as depicted in this study. Previous studies also revealed that the CMR is high in SCD patients compared with healthy peer groups and could be used as an indicator for the extent of dehydration.^[21,22] In agreement with the facts, the present study observed low serum calcium, magnesium, and raised phosphorus levels and accordingly low CPR and high CMR in the crisis group as compared with the steady state group [Table 4] Low serum calcium recorded a significant AUC as predictor for crisis state [Table 6] Serum calcium value less than 2.41 mmol/L (9.65 mg/dL) showed a sensitivity of 83.3% for crisis [Table 6 and Figure 3]. The observations suggested that these changes could be due to the shift of extracellular Ca²⁺ into the red cells during the crisis phase. Hence, treatment using calcium channel inhibitors and oral magnesium supplementation might be beneficial in preventing crisis episodes.

Serum albumin is generally considered a marker of the individual’s nutritional status. Besides, it is the primary determinant for the plasma oncotic pressure and, thus, the key determining factor for the fluid distribution between the extra- and intravascular compartments.^[23] Low serum albumin levels correspond to low fluid holding capacity in the intravascular space, resulting in altered plasma osmotic pressure and osmolality. Plasma albumin is also attributed to RBC membrane stabilization and the shape and volume of RBCs.^[8,24,25] Low albumin also depicted a significant inverse relation with the disease severity score [Table 5]. A cutoff value of 0.63 mmol/L (4.2 g/dL) showed a sensitivity of 88.9% for the crisis state [Table 6 and Figure 3]. Although the cations and anions in the crisis state were significantly decreased [Table 4], the ratios of serum osmolality with these variables were not different between the groups. Of all the ratios of osmolality with serum parameters, SAR and OABR reported a significant difference between the crisis and steady state groups [Table 4]. The sensitivity for a patient to have a crisis event was nearly 94.4% if the SAR values were higher than 208.45 [Table 6 and Figure 2]. Further, it was observed that an OAR value greater than 433.79 also depicted similar sensitivity of 94.4% (data not shown). Hence, the contribution of serum albumin toward fluid homeostasis and red cell membrane stability cannot be ignored. The findings agree with the Nouraie et al study, which observed a significant association of low serum albumin with higher mortality and proposed that albumin could be used as a biomarker for early assessment of clinical severity in SCD cases.^[26] Furthermore, it was observed that in all the enrolled steady state cases [Figure 1] the levels of serum sodium, calcium, magnesium, osmolality, and albumin were within the normal reference range. None had low levels of these parameters. The key note of the present study is that prevention of red cell dehydration is one of the crucial factors for reducing the frequency of vaso-occlusive events. Hence, water and electrolyte balance should be considered for crisis prevention and management in these patients.

The study has some limitations. The small sample size is one of the significant limitations. A prospective longitudinal study including a larger cohort of HbSS cases that could be followed up regularly could provide a better understanding of the changes in serum ions and their influence on SCD pathophysiology. Instead of measuring total serum calcium and magnesium, ionized calcium and magnesium would have explored a more precise relationship with the disease severity in these patients. The study subjects’ nutritional status and total body water content were not assessed in this study, which could have provided better insight regarding the low serum albumin and magnesium. However, the advantage of the study is that it is a very comprehensive study that included the major serum cations and anions, including osmolality in the study groups, to elicit the prognostic impact of these parameters on disease severity. We could find very few studies that investigated all these serum variables in SCD cases. In addition, comparing the values with the healthy control group and between the crisis and steady state groups provided better insight into the changes occurring during the crisis. Further, additional research is necessary to determine the exact mechanism of the changes occurring in the microvasculature of SCD patients.

CONCLUSIONS

The present study confirmed that low serum sodium, calcium, and albumin were significantly associated with crisis events in SCD cases. In addition, raised SAR and OAR were also linked to the crisis state, and the resultant effects of the shift of ions between the compartments might be a key factor for intracellular dehydration and autopolymerization of HbS. Hence, routine estimation of these ions will help in the early assessment of the clinical severity. These parameters can be easily measured at any rural or urban health care center. Accordingly, SCD patients could be advised to proper fluid intake with adequate electrolyte and nutrient supplementations to maintain fluid homeostasis and prevent the frequency of crisis episodes.

HIGHLIGHTS

• The major serum electrolytes and osmolality are reduced in SCD cases than their contemporary healthy individuals. Serum cations like sodium, calcium, and magnesium, and anions like chloride and albumin are decreased in the SCD crisis state resulting in disproportionate distribution of ions across the red cell membrane.

• Low calcium, albumin, and sodium–albumin ratio are significantly associated with disease severity score and better sensitivity.

• Routine estimation of these ions will help in the early assessment of clinical severity.

• Adequate fluid and electrolyte intake would help maintain fluid homeostasis and prevent the frequency of crisis episodes in SCD cases.