Total Serum Calcium Level Is Negatively Correlated With Systemic Lupus Erythematosus Activity.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease and hypocomplementemia signifies disease activity. Several studies have shown that calcium may help maintain optimum function of immune system and metabolism in SLE. The aim of our study was to analyze the relationship between total serum calcium level and SLE activity. A total of 66 patients with SLE and 214 healthy controls were included in this study. Our results showed lower serum levels of calcium (P < .001), complement C3 (P < .001), complement C4 (P < .001), and albumin (P < .001) in patients with SLE. A negative correlation was found between serum calcium level and systemic lupus erythematosus disease activity index (SLEDAI) rating (r = -0.394, P = .001). Additionally, serum level of calcium was positively correlated with serum complement C3 level (r = 0.366, P = .003) in patients with SLE, while no such correlation was found between serum calcium level and complement C4 (r = -0.190, P = .126). Likewise, patients with SLE with normal serum calcium level showed higher complement C3 level (P < .01) than that of patients with low serum calcium level. Overall, the results displayed that patients with SLE have lower serum calcium level compared to healthy controls, and the serum calcium level is positively correlated with SLEDAI rating and serum complement C3 level in patients with SLE. In conclusion, the total serum calcium level is negatively correlated with SLE disease activity.

Introduction

Systemic lupus erythematosus (SLE) is a systemic chronic autoimmune disease across the world among different ethnic and racial groups. Systemic lupus erythematosus develops predominantly in women of childbearing age and is more prevalent in non-Caucasians.[1] Genetic predisposition, environmental susceptibility, and hormonal homeostasis contribute to disease development and activity.[2] Clinical manifestations and the pattern of organ involvement are widely heterogeneous among patients with SLE, reflecting the complex pathogenic mechanisms of SLE. Notably, complement involvement and activation play an important role in the inflammatory response prompted by immune complex deposition in autoimmune-mediated tissue injury. According to the validated criteria published by Systemic Lupus International Collaborating Clinics (SLICC) group, low serum levels of complement C3 and C4 are included as one of the immunological classification criteria of SLE.[3-5] The significance of complement to reflect SLE activity has been confirmed.[6] Low serum complement C3 level was regarded as the most significant risk factor for cytopenia and mucocutaneous lesions and was negatively correlated with SLE disease activity.[7]

It is reported that ionized calcium (Ca2+) plays a key role in both intra- and extracellular compartments.[8] A recent study found that total serum calcium level may have adverse effects on serum cholesterol and triglycerides[9] and hypertension.[10] Calcium signaling participates in many pathways involving in immune-tolerance and inflammation. For instance, Ca2+signals were demonstrated to be involved in B cell development and fate, which were key aspects of immune tolerance.[11] Moreover, recent studies suggested that calcium signaling could regulate cyclic GMP-AMP synthase/stimulator of interferon genes axis activation, thus participating in the innate immunity modulation through type I interferons.[12] Due to its effect on the innate and adaptive immunity, abnormal calcium signaling was associated with several autoimmune diseases including SLE.[12,13] Studies displayed that calcium responses in SLE lymphocytes were increased following the participation of the antigen receptor. In T cells from patients with SLE, engagement of the T cell receptor results in increased production of inositol 1,4,5-trisphosphate (IP3) and increased release of calcium from endoplasmic reticulum.[14] Likely, the B cell receptor–mediated calcium response is also increased.[15] However, the clinical value of serum calcium level in patients with SLE has not been explored yet.

Calcium homeostasis is maintained and regulated by 1,25(OH)2D3, which is the active form of vitamin D.[16] It is reported that vitamin D deficiency is common in patients with SLE,[17]and vitamin D supplementation is of benefit in modulating disease activity. Considering the important role of calcium signaling in autoimmune diseases and the clinical significance of vitamin D supplementation in SLE, the change of serum calcium level in patients with SLE and its clinical value should be investigated further. Therefore, this study was carried out to evaluate the serum calcium level between the patients with SLE and normal controls and to investigate the correlation between total serum calcium level and SLE disease activity.

Material and Methods

Patients

A total of 66 patients with SLE (54 females, 12 males, mean age: 49.00 ± 14.72) and 214 age- and gender-matched healthy controls (40 females, 174 males, mean age: 52.34 ± 14.85) were randomly selected from the individuals who received clinical laboratory analysis in Liyang People’s Hospital from 2016 to 2019 after obtaining their informed consent. The patients with SLE were diagnosed according to the revised criteria of the American College of Rheumatology.[18,19] Patients did not receive any drugs and their diseases were in the active state. The evaluation of diagnosis, disease activity, and clinical data acquisition were performed by the same physician. The SLE disease activity index (SLEDAI) rating system was applied to assess SLE disease activity. The ratings were as follows:[20] 0 to 9 for the slight activity of patients with SLE, 10 to 14 for the moderate activity of patients with SLE, and ≥15 for high activity of patients with SLE. Physical examination data including age, gender, height, weight, and body mass index (BMI) were obtained from both the study groups (SLE group and control group). Clinical information of patients with SLE including a family history of autoimmune diseases, duration of the disease, smoke, alcohol consumption, lymphadenopathy, skin lesions, arthritis, nephropathy, cardiac, or central nervous system (CNS) involvement and laboratory reports about anticardiolipin antibody, anti-dsDNA antibody, antinuclear antibody, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level was recorded on a prescribed pro forma.

For the healthy controls, subjects with any history of autoimmune inflammatory diseases, recent infection, and iron-deficiency anemia were excluded from this study. The study was performed in accordance with the ethical standard in the Declaration of Helsinki and was approved by the institutional research board of Nanjing Medical University. The clinical and laboratory findings of the patients are given in Tables 1 and 2.

Table 1.

General Characteristics of Patients With SLE and Healthy controls.a

Parameters	SLE (n = 66)	Controls (n = 214)	P value
Age (years)	51.58 ± 14.42	55.58 ± 16.03	.070
Gender (male/female)	12 (18)/54 (82)	40 (19)/174 (81)	.926
Height (m)	1.61 ± 0.07	1.62 ± 0.07	.245
Weight (kg)	53.85 ± 6.93	54.24 ± 7.65	.712
BMI (kg/m2)	20.80 ± 1.78	20.62 ± 1.65	.444
Smoke (yes/no)	15 (23)/51 (77)	38 (18)/176 (82)	.368
Alcohol consumption (yes/no)	8 (12)/58 (88)	23 (11)/191 (89)	.756

Abbreviations: BMI, body mass index; SLE, systemic lupus erythematosus.

a Measurement data are expressed as mean ± SD and categorical variables are expressed as frequency/percentage, each for the patients and controls.

Table 2.

Clinical Characteristics of Patients With SLE Included in This Study.

Characteristics	Categories	Patients with SLE (n = 66)Frequency/percentage
ESR (mm/h)	≤20 mm/h	2 (3)
	>20 mm/h	64 (97)
CRP (mg/L)	≤5 mg/L	17 (26)
	>5 mg/L	49 (74)
SLEDAI rating	Slight activity (0-9)	41 (62)
	Moderate activity (10-14)	23 (35)
	High activity (≥15)	2 (3)
Family history of autoimmune disease	Yes	5 (8)
	No	61 (92)
Duration (months)	1-5 year	18 (27)
	>5 years	48 (73)
Lymphadenopathy	Yes	45 (68)
	No	21 (32)
Skin lesions	Yes	39 (59)
	No	27 (41)
Arthritis/arthralgia	Yes	14 (21)
	No	52 (79)
Cardiac involvement	Yes	11 (17)
	No	55 (83)
CNS involvement	Yes	17 (26)
	No	49 (74)
Nephropathy	Yes	53 (80)
	No	13 (20)
Anticardiolipin antibody	Positive	21 (32)
	Negative	45 (68)
Anti-dsDNA antibody	Positive	52 (79)
	Negative	14 (21)
Antinuclear antibody	Positive	63 (95)
	Negative	3 (5)

Abbreviations: CNS, central nervous system; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; SLEDAI, systemic lupus erythematosus disease activity index.

Serum Samples Collection and Laboratory Assays

After obtaining informed consent from the subjects, blood samples were withdrawn aseptically from patients with SLE and healthy individuals without venous stasis and frothing. The samples were then centrifuged at 3000 rpm for 10 minutes. Serum samples were separated and shifted into properly labeled tubes. The tubes were then restored at −20 °C for further analysis. Complement C3, complement C4, and albumin were measured by latex-enhanced Behring nephelometry with high-sensitivity assays (BN100 nephelometer; Dade Behring). The normal ranges of complement C3, complement C4, and albumin were 0.88 to 2.0, 0.16 to 0.47, and 35 to 55 g/L, respectively. Total serum calcium level was measured by Hitachi 7020 analyzer and the normal range was 2.1 to 2.8 mmol/L.

Statistical Analysis

All the statistical analysis were done by the statistical software SPSS 13.0 (SPSS, Inc.). The data were presented as the mean ± SD.

Student unpaired t test was applied to compare the difference of serum parameters between the patients with SLE and healthy individuals. χ2 test was used to compare the clinical distribution between patients with SLE and healthy controls. The correlation of total serum calcium level with SLEDAI rating, complement C3, complement C4 and albumin, was analyzed by Pearson partial correlation coefficient. All statistical tests were 2-sided, and the value of P < .05 was considered as a statistically significant.

Results

General Characteristics

In the present study, a total of 280 subjects were recruited for the investigation. No significant differences for age, gender, height, weight, and BMI were found between patients with SLE and healthy controls. Systemic lupus erythematosus was more prevalent in female with male to female ratio of 1:4 approximately. The demographic distribution of smoker and alcohol consumer displayed no difference between patients with SLE and healthy controls (Table 1). In this study, the persistence of the disease was 1 to 5 years in 27% of the patients with SLE and was more than 5 years among most of the patients (73%). Most of the patients with SLE showed elevated ESR (98%) and increased level of CRP (74%). Sixty-two percent of patients displayed the slight activity of SLE according to SLEDAI rating system, while patients showing moderate activity and high activity of SLE accounted for 35% and 3%, respectively. Eight percent of the patients with SLE had a family history of autoimmune diseases. There were 68% patients showing lymphadenopathy, 59% showing skin lesions, 21% showing arthritis, 80% showing nephropathy, 17% showing cardiac involvement, and 26% showing CNS involvement. Additionally, anticardiolipin antibody was positive in 32% patients while it was negative in 68% of the patients. Anti-dsDNA antibody and antinuclear antibody were positive in 79% and 95% of the patients, respectively (Table 2).

Changes in Complement C3, Complement C4, and Total Serum Calcium Level in Patients With SLE

The patients with SLE had significantly lower serum level of complement C3 (P < .001), complement C4 (P < .001), and albumin (P < .001) than healthy individuals. Notably, the total serum level of calcium was found significantly lower in patients with SLE (P < .001) than the control group (Table 3). Moreover, a stratified analysis of serum calcium level was performed according to gender and age to explore the difference further. Notably, both male and female patients with SLE showed significantly lower serum calcium level than that of healthy controls and such difference could be found in all ages as well (Table 4). Furthermore, stratified analysis of serum calcium level among patients with SLE displayed that younger female patients (<60 years old) displayed relatively higher serum calcium level than male patients with SLE (P < .001) and older female patients (60 years old or older, P = .041; Table 5).

Table 3.

Laboratory Analysis of Serum Complement C3, Complement C4, Total Calcium, and Albumin Level Between Patients With SLE and Healthy Controls.

Parameters	SLE (n = 66)	Controls (n = 214)	P value
Complement C3 (g/L)	0.65 ± 0.22	0.90 ± 0.29	.000a
Complement C4 (g/L)	0.16 ± 0.07	0.23 ± 0.07	.000a
Serum calcium (mmol/L)	2.13 ± 0.21	2.26 ± 0.14	.000a
Albumin (g/L)	34.04 ± 6.25	38.57 ± 5.06	.000a

Abbreviation: SLE, systemic lupus erythematosus.

a P < .001.

Table 4.

Stratified Analysis of Serum Calcium Level Betweens Patients With SLE and Healthy Controls by Gender and Age.

Gender and age	Calcium (µmol/L)				P value
	Patients with SLE (n = 66)		Controls (n =214)
	Mean	SD	Mean	SD
Male
<60 years	1.89	0.18	2.27	0.13	.000a
≥60 years	1.99	0.14	2.30	0.15	.000a
Female
<60 years	2.20	0.20	2.27	0.13	.016b
≥60 years	2.07	0.16	2.24	0.15	.000a

Abbreviation: SLE, systemic lupus erythematosus.

a P < .001.

b P < .05.

Table 5.

Stratified Analysis of Serum Calcium Level Among Patients With SLE by Gender and Age.

Gender and age	Calcium (µmol/L) level in patients with SLE (n = 66)		P value
	Male (n = 12)	Female (n = 54)
<60 years (n = 50)	1.89 ± 0.18	2.20 ± 0.20	.000a
≥60 years (n = 16)	1.99 ± 0.14	2.07 ± 0.16	.391
P value	0.310	0.041b

Abbreviation: SLE, systemic lupus erythematosus.

a P < .001.

b P < .05.

Correlation Between Total Serum Calcium Level and Disease Activity and Complement C3 and Complement C4

To explore the correlation between total serum calcium level and SLE activity, Pearson partial correlation coefficient was conducted between serum calcium level and SLEDAI rating, serum level of complement C3 and complement C4, and albumin. The results showed that serum calcium level was negatively correlated with SLEDAI rating (r = −0.394, P = .001), indicating that lower serum calcium level might signify higher activity of the disease. Total serum calcium level was positively correlated with serum level of complement C3 (r = 0.366, P = .003) in patients with SLE while such correlation was not found between total serum calcium level and complement C4 (r = −0.190, P = .126; Table 6). No correlations were found between total serum calcium level and complement C3 and complement C4 in healthy controls (Table 6). As majority of serum calcium circulates and binds to albumin,[21] serum albumin level was measured and was positively correlated with total serum calcium level both in patients with SLE (r = 0.773, P < .001) and in healthy controls (r = 0.541, P < .001).

Table 6.

Correlation Between Serum Calcium Level and Complement C3 and Complement C4.

Parameters	SLE (n = 66)		Controls (n = 214)
	R	P	r	P
SLEDAI rating	−0.394	.001a	-	-
Complement C3 (g/L)	0.366	.003a	0.082	.233
Complement C4 (g/L)	−0.190	.126	0.011	.871
Albumin (g/L)	0.773	.000b	0.541	.000b

Abbreviations: r, Pearson correlation coefficients; SLEDAI, systemic lupus erythematosus disease activity index.

a P < .01.

b P < .001.

To further investigate the relationship between serum calcium level and complement C3, patients with SLE and healthy controls were divided into 2 groups according to their serum calcium level. Patients with SLE who had the serum calcium level lower than 2.1 mmol/L were regarded as the patients with low serum calcium level and patients with serum calcium level ranging from 2.1 to 2.8 mmol/L were classified as patients with normal calcium level. Notably, patients with SLE with normal calcium level showed significantly higher serum level of complement C3 than that of patients with lower serum calcium level (P < .01). Consistent with the previous results, no such correlation was found in healthy controls (Figure 1).

Figure 1.

Mean serum level of complement C3 according to total serum calcium level in patients with SLE and healthy controls. SLE indicates systemic lupus erythematosus.

Discussion

Systemic lupus erythematosus is one of the most quintessential autoimmune diseases. The clinical manifestations and autoimmune phenomena are heterogenous among patients with SLE and even changes over time in individuals. Therefore, more systematic tests should be applied to improve the sensitivity and specificity for SLE diagnosis. The Systemic Lupus International Collaborating Clinics group put forward that hypocomplementemia involving C3, C4, and total hemolytic complement (CH50) could be an immunologic marker to enhance the sensitivity of the SLE classification criteria in 2009.[5] Lower serum complement C3 and C4 levels were confirmed to signify active SLE by lots of studies.[3,22-24] Although hypocomplementemia has important value for SLE activity assessment, it was also highly prevalent in other diseases. Therefore, low serum complement C3 or C4 level alone may be not qualified to assess the disease activity and more correlated parameters should be investigated to improve the sensitivity and specificity of SLE activity assessment.

Vitamin D deficiency was found to be common in patients with SLE.[17] Vitamin D receptor (VDR) polymorphism, which was associated with lower serum level of vitamin D, has been found to be associated with the incidence of SLE as well.[25] Furthermore, vitamin D deficiency was found to be correlated with SLE activity,[26-28] making vitamin D supplementation necessary in all patients with SLE with insufficiency or deficiency for its immunomodulatory effects. In the longitudinal observational study conducted by Petri et al,[29] vitamin D supplementation aimed to a 25(OH) vitamin D level of 40 ng/mL was associated with a reduction of proteinuria and higher complement C3 and C4 levels. Recent studies also found that lower levels of vitamin D were associated with more consumption of complement C3 and complement C4 and higher disease activity rate.[30] Furthermore, VDR messenger RNA level was shown to be positively associated with serum complement C3.[31] Calcium homeostasis is maintained and regulated by 1,25(OH)2D3, the active form of vitamin D and serum calcium is commonly measured in laboratory examination. Therefore, due to the clinical significance of serum 1,25(OH)2D3 level and vitamin D supplementation in SLE, the change of serum calcium level and its clinical value should be explored and characterized. The present study suggested that patients with SLE had lower serum calcium level than that of healthy controls (P < .001). Notably, it was the first time to show that serum calcium level is related to SLEDAI rating (P = .001) and serum complement C3 level (P = .003) in patients with SLE, suggesting that total serum calcium level is correlated with SLE activity. As low serum calcium level could be treated by high-calcium food intake and vitamin D supplementation,[32] our findings might be of clinical significance in SLE modulation and treatment.

Calcium circulates in the blood in 3 forms. Free ionized calcium (Ca2+) accounts for approximately 50%, with the remainder 40% bound to albumin and 10% forming complex with anions such as phosphate, bicarbonate, and lactate.[21] Despite that Ca2+ is the physiologically active form of calcium, clinical laboratories routinely measure total serum calcium level. Notably, the serum albumin level in patients with SLE was significantly lower than that in healthy controls (P < .001). Therefore, the change of total serum calcium level in patients with SLE in our study could partly be attributed to the decrease of serum albumin level and whether the change in serum ionized calcium level remained unknown. Further studies should be conducted to investigate the change of different forms of calcium in patients with SLE and to disclose the underlying mechanism of how SLE disease activity influence calcium homeostasis.

This study also has several limitations: To begin, our study included a smaller sample size. Second, it is reported that serum iron level is negatively correlated with serum triglyceride,[33] and free fatty acids are associated with SLE.[34] Therefore, whether other trace elements are associated with SLE activity and prognosis should be explored further. Furthermore, double-blinded randomized clinical trials should be applied to confirm the cause and effect relationship between serum level of calcium and disease activity. Other dietary habits and lifestyles also should be thought in the future study. For example, some study found obesity increase in the incidence of new-onset lupus nephritis. However, the previous study indicated that the prevalence of obesity is high in China in different population.[35-37]

In conclusion, these findings suggest that patients with SLE have lower serum calcium level compared to healthy controls and serum calcium level is negatively correlated with SLE activity.