Systemic
lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by
immune dysregulation, which results in the production of autoantibodies,
activation of the complement system, and generation of immune complexes [
In several
past decades, the use of corticosteroids and cytotoxics has dramatically
improved the prognosis of the disease [
The aim of this study was to evaluate the frequency of gallstone formation and alteration of plasma lipid profiles in SLE patients treated long with prednisolone.
This was a
retrospective study covering 60 consecutive SLE patients (45 females and 15
males; Caucasians; mean age
Baseline disease-related features of the SLE patients recruited.
Variable | |
---|---|
≥4 ACR criteria, | 60 (100) |
Sex (female/male) | 44/16 |
Median age at the time of this study, | 28 (18–44) |
Median age at the onset of disease,
| 20 (10–36) |
Median age at diagnosis, | 21 (15–29) |
Median disease duration, | 9.6 (1–21) |
Median (IQR) SLEDAI, score | 15 (10–22) |
Median (IQR) SLICCA damage index, score | 1.1 (0.4–2.3) |
Current steroid therapy, | 32 (53.3) |
Median (IQR) current steroid dosage, mg | 5.5 (0–15.5) |
Median (IQR) steroid therapy duration, years | 5 (0–12) |
Median (IQR) monthly steroid dose, mg/month | 12 (0–16) |
Cumulative steroid dose (range), g | 107.3 (0–583.7) |
Current antimalarial therapy, | 24 (40) |
Current immunosuppressive therapy,
| 16 (26.7) |
Gallstone disease, | 22 (36.7) |
IQR: Interquartile range.
The mean
monthly and cumulative dose of prednisolone and its treatment duration were recorded during a chart review.
The mean monthly dose of prednisolone was defined as its total sum in
milligrams divided by the total number of follow-up months, the cumulative dose
of the agent was as its total sum in grams taken during the same period, and
the duration of treatment with the drug was estimated as the time interval
(years) during which a patient had taken it (Table
SLE activity
was quantified, by using the SLE disease activity index (SLEDAI) [
The patients
were clinically interviewed and examined according to the standard protocol
comprising demographic characteristics, family history, and lifestyle (Table
Comparisons of baseline demographic characteristics, prednisolone therapy, SLE-related features, and laboratory data in SLE patients with/without gallstones.
Variable | SLE patients with gallstones
( |
SLE patients without gallstones ( | |
---|---|---|---|
Sex (female/male) | 17/5 | 27/11 | NS ( |
Median age at the time of this study (range), years | 33 (22–44) | 24 (18–40) | .0002 |
Median age at the onset of the disease (range), years | 23 (10–35) | 19 (8–36) | .015 |
Median age at diagnosis (range), years | 25 (18–35) | 21 (17–37) | NS |
Median disease duration (range), years | 11.8 (8–15) | 1.8 (0–8.0) | .000001 |
Median (IQR) SLEDAI, score | 7 (6–10) | 12 (10–20) | .000145 |
Median (IQR) current steroid dosage, mg | 16.0 (10.0–18.5) | 2.1 (0–5.0) | .0001 |
Median (IQR) steroid therapy duration, years | 12 (8.0–15.0) | 0 (0–5.0) | .0001 |
Median (IQR) monthly steroid dose, mg/month | 16.1 (15.0–16.5) | 0 (0–8.0) | .0001 |
Cumulative steroid dose (range), g | 848.0 (502.0–972.5) | 0 (0–65.7) | .0001 |
TCh (range), mg/dL | 220.0 (172.0–251.0) | 177.0 (142.0–190.0) | .0004 |
HDL-Ch (range), mg/dL | 46.0 (40.0–56.0) | 38.0 (34.0–42.0) | .008 |
Ratio of TCh and HDL-Ch | 4.9 (4.1–5.5) | 4.3 (3.6–4.8) | .05 |
LDL-Ch (range), mg/dL | 135.0 (103.0–173.0) | 111.2 (84.6–136.0) | .006 |
ApoA1-HDL (range), mg/dL | 130.0 (115.0–151.0) | 112.0 (103.0–141.0) | .05 |
ApoB-LDL (range), mg/dL | 108.0 (89.0–136.0) | 97.0 (77.0–119.0) | .005 |
Fibrinogen (range), g/L | 3.8 (3.1–4.2) | 3.4 (2.6–4.2) | NS |
TCh: Total cholesterol; HDL-Ch: HDL
cholesterol; LDL-Ch: LDL cholesterol; IQR: Interquartile range; NS: Nonsignificant.
All comparisons are Mann-Whitney
All samples
were collected after a written informed consent had been given under
institutional review board-approved protocols in accordance with the Helsinki
Declaration. Hemoglobin, leukocyte and platelet counts, fasting plasma glucose,
serum creatinine, serum liver functional tests (bilirubin, cholesterol,
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase,
total protein, albumin, and globulin levels), urine microscopy, DNA antibodies,
complement profile (
Ultrasonography was performed with a Combison 530 with 3.5 MHz convex (Kretz, Austria). Gel was applied to the skin to provide an acoustic interface.
Blood was sampled after a 14-hour of fasting. On the day of sampling, plasma and
serum were immediately separated, centrifuged, and stored at −20°C before
testing the lipid profile. TCh,
TG, and HDL-Ch were measured, by employing commercially available assays (Biocon,
Germany) on an Airone-200 analyzer (Crony, Italy). HDL-Ch
was measured after precipitation of LDL and very low-density lipoprotein
(VLDL), by using sodium 12-tungstophosphate (1.1 mmol/L) and magnesium chloride
(45 mmol/L) (a final concentration) [
Color
cathodoluminescence scanning electron microscopy (CCL-SEM) was employed to
study the composition and structure of gallstones. A “Stereoscan MK-IIA” SEM
(Cambridge Instruments, UK) was equipped with a CCL-attachment (developed by
GVS, SKO, and PVI at the Department of
Physics, Moscow State University, Russia) and operated in the “real-color mode”
at an electron energy of under 20 keV and a beam current of 10 nA. As a result,
color images of the samples under examination were electron-induced luminescence
of major chemical gallstone components located onto the section surface. “Adobe
Photoshop” software was used to separate colors and to have intensity
histograms. The method and its possible fields of application were
characterized in detail in our earlier publications [
The standard
commercial unconjugated bilirubin (
CCL-SEM micrographs of bilirubin (a), cholesterol (b), high molecular weight protein (c) and their corresponding histograms obtained after color separation.
The results
for continuous variables were expressed as mean ± SD. Nonparametric tests were carried
out due to the skewed distribution of the Gaussian curve and the small number
of samples. Differences between groups were examined by the Mann-Whitney
Univariable association of predisposing factors to gallstone formations in the study SLE patients.
Variable | SLE patients with | SLE patients without | OR | Ln OR | SE ln OR | 95% CI | |||
---|---|---|---|---|---|---|---|---|---|
gallstone ( | gallstone ( | ||||||||
present, | not, | present, | not, | ||||||
Age at the onset of the disease (≥25.1 years) | |||||||||
Age at the time of this study (≥30.3 years) | |||||||||
Prednisolone therapy duration (≥7.0 months) | |||||||||
Current steroid dosage (≥8.66 mg.) | |||||||||
Minimum maintenance dosage of prednisolone (≥10.2 mg) | |||||||||
Maximum maintenance dosage of prednisolone (≥28.5 mg) | |||||||||
Monthly steroid dose (≥10.4 mg) | |||||||||
Cumulative steroid dose (≥498.5 mg) | |||||||||
Pregnancy in past history | |||||||||
Enanthema of mucosal soft palate | |||||||||
Thrombocytopenia | |||||||||
Hemolytic anemia | |||||||||
Central nervous system lesion | |||||||||
Lupus nephritis | |||||||||
IgG-anticardiolipin antibody (≤11.6) | |||||||||
Antiphospholipid syndrome | |||||||||
TCh (≥169.0 mg/dL) | |||||||||
HDL-Ch (≤45.5 mg/dL) | |||||||||
ApoB-LDL(≤74.0 mg/dL) | |||||||||
LDL-Ch (≤136.0 mg/dL) | |||||||||
ApoA1-HDL (≥116.5 mg/dL) | |||||||||
Ratio of TCh and HDL-Ch (>4.5) | |||||||||
Ratio of HDL-Ch and HDL-PhL (≥0.504) |
TCh: Total cholesterol; HDL-Ch: HDL cholesterol; LDL-Ch: LDL cholesterol; HDL-PhL: HDL phospholipids.
Table
To evaluate the impact of various factors on
the development of cholelithiasis in SLE patients, all clinical signs and
laboratory data were compared in the gallstone and nongallstone groups. Table
The signs showing the significance of group
differences of less than 0.05 were regarded as risk factors for gallstone
disease in SLE patients. For univariate analysis, the most informative signs were
chosen from all the variables
included into the study and found to differ significantly. The frequency of
this variable (present, absent) in a group, odds ratio (OR), natural logarithm
of the odds ratio (ln OR), standard error in the odds ratio (SE ln OR), 95%
confidence interval (CI) for the odds ratio, and significance P by the
The data given in Table
The values of
the study variables at
Table
The predisposing factors to gallstone formation in the study SLE patients.
Variable | The Fisher exact test | |
---|---|---|
Age at the onset of the disease | 93.8 | <.000001 |
Current steroid dosage | 109.2 | <.0000001 |
Steroid therapy duration | 152.0 | <.0000001 |
As we
expected, the levels of plasma lipids, ApoB-LDL (
When the
group of SLE patients treated with corticosteroids were additionally compared,
significantly higher levels of TCh (
Interestingly, the levels TG and ApoB-LDL, as well as TCh/HDL-Ch and LDL-Ch/HDL-Ch ratios, did not differ significantly in corticosteroid-treated SLE patients as compared with patients who had never been treated with corticosteroids.
Series of
CCL-SEM color images of 10 gallstones were taken for analysis. Each image was
compared with control images to distinguish the deposition of bilirubin,
cholesterol, and protein components. The major components of the gallstones
under examination were cholesterol (Figure
CCL-SEM micrographs demonstrating the structural organization and chemical composition of a gallstone obtained from SLE patient: (A) general gallstone structure before color separation; (a) bilirubin inclusions; (b) cholesterol component; (c) protein.
Gallstone disease is one the most common disorders of the digestive system. In several past decades, there has been a tendency toward the increased incidence of gallstone disease. It can be explained by better diagnosis and by the new noninvasive ultrasound techniques being introduced into clinical practice.
Any publications on the incidence and mechanisms of cholelithiasis in SLE have not been found in the literature. This study has examined the relationship between gallstone disease and corticosteroid treatment in SLE. As far as we know, this is the first study to show the high frequency of gallstone disease in SLE patients treated with prednisolone. Regression and discrimination analyses reveal a strong association of gallstone formation in SLE patients with age at the onset of the disease, current steroid dosage, and duration of prednisolone therapy. Cholesterol metabolic disturbance is one of the possible causes of gallstone formation in SLE patients on prednisolone therapy.
Since there
is a strong correlation between cholelithiasis and blood lipid-protein spectral
changes in patients on corticosteroid therapy, we hypothesize that cholesterol
metabolism occurring on
Our findings suggest
that higher total cholesterol levels in SLE patients with gallstones are most
likely to be caused by cholesterol catabolic disorders. Up to 80% of the total
cholesterol pool is oxidized to bile acids [
The effects
of corticosteroids on the lipid-protein blood spectrum have been studied
before. The plasma lipid abnormalities reported in SLE corticosteroid treatment
patients are similar to those in other dyslipoproteinemias [
Our SLE
patients with gallstones had significantly elevated LDL-Ch and HDL-Ch levels,
as compared to those without gallstones (Table
Recent
advances in bile molecular biology and biochemistry have demonstrated that
gallstone disease is a complex disorder: cholesterol supersaturation, decreased
bile acid/cholesterol ratio, hydrophobic bile salts, pronucleating proteins,
inflammation changes, and impaired gallbladder motility [
Steroid hormone catabolism also takes
place in the hepatocytes through the
We assume
that prednisolone competitively inhibits hepatocytic alpha-hydroxylases and
thus decelerates cholesterol transformation into bile acids. Subsequently, it
raises the serum levels of total cholesterol, LDL-Ch, and HDL-Ch, and also
increases cholesterol excretion into bile. Simultaneously, enhanced cholesterol
excretion is accompanied with the reduced hepatocytic secretion of bile acids
into the bile capillaries, resulting in a decrease in the bile acid/cholesterol
ratio. In SLE patients receiving long steroid hormones, gallbladder
inflammatory changes and immunological changes in the presence of abnormal
cholesterol and protein metabolisms may initiate gallstone formation (Figure
Diagram of prednisolone (steroid hormones)-induced inhibition of cholesterol catabolism (see the text for comments).
Cholesterol
gallstones are more common in women than in men, and exposure to oral
contraceptive steroids and conjugated estrogens increases the risk for
gallstones [
There are
also reports on that high biliary protein concentration in lithogenic bile was associated with
cholesterol gallstone formation [
Despite
the importance of high protein and cholesterol concentrations on the sequence
of events in gallstone formation, very little is known about the relationship
between cholesterol and protein to the
microarchitectonics of gallstones [
In present study, we used the novel electron microscopy technique, color cathodoluminescence scanning electron microscopy (CCL-SEM), to investigate the chemical composition and microstructure of gallstones obtained from patients with SLE.
The main advantage of CCL-SEM over SEM is its capability to
visualize different organic and inorganic components and their distribution
over the gallstone section surface [
The CCL-SEM technology allowed us to generate CCL-mapping of
color images of 3 constituents of studied gallstones, unesterified cholesterol,
unconjugated bilirubin, and protein (Figure
In conclusion, gallstone disease was detected in 36.7% of the examinees; 68.8% of SLE patients on corticosteroid therapy had cholelithiasis. Age at the onset of the disease, current steroid dosage, and steroid therapy duration are factors that predispose to gallstone formation. The CCL-SEM study identified predominantly the protein-cholesterol structure of gallstones. Multicenter-randomized studies are required to support the aforesaid assumption that long-term steroid hormones therapy is a model of cholesterol lithogenesis. Moreover, if there is evidence that cholelitiasis is induced by steroids, this will provide impetus to the search for and design of new drugs and therapeutic approaches to treating autoimmune diseases.
The authors would like to thank Professor Nataliya V. Perova, Dr. Irina N. Ozerova (State Research Center for Preventive Medicine, Moscow, Russia) for obtaining the data on the lipid profile, and Dr. Tatiana B. Prokaeva (Boston University School of Medicine, Boston, Mass, USA) for their assistance and support of this work. They also would like to thank Aleksandr K. Tarasov, Candidate of Engineering Sciences, and Mikhail I. Barsov for assistance in the statistical processing of the findings.