Is there a relationship between lipids metabolism and splenic surgeries ?

PURPOSE: To assess the influence of spleen surgeries (splenectomy, presence of spleen and after conservative surgeries) on lipids metabolism. METHODS: Fifty female Wistar rats of similar weight and age were divided into five groups submitted to the following procedures: Group 1 control, with an intact spleen; Group 2 sham operation, Group 3 – total splenectomy; Group 4 subtotal splenectomy, and Group 5 total splenectomy complemented with autogenous spleen tissue implants. Four months after the interventions, serum triglycerides, total cholesterol and fractions (VLDL-cholesterol, LDL-cholesterol, HDL-cholesterol) were determined. The results for the four groups were compared by analysis of variance followed by the Tukey-Kramer test, with the level of significance set at p<0.05. RESULTS: There were no differences between groups 1, 2, 4 and 5. In the animals submitted to total splenectomy, total cholesterol (p=0.0151) and LDL-cholesterol fraction concentrations (p<0.0001) were higher, whereas HDL-cholesterol fraction concentrations were lower (p=0.0026) than those detected in the other groups. There was no difference in triglycerides (p=0.1571) or VLDL-cholesterol (p=0.2527) between groups. CONCLUSION: Splenectomy is related to changes in the lipid metabolism that are reverted by autogenous spleen tissue implants.


Introduction
The functions of the spleen are part of the organic metabolism and disorders of this organ are related to different diseases [1][2][3][4][5][6][7][8][9][10] .The lysosome deposits are considered to be the most frequent metabolic changes involving the spleen associated with an enzymatic defect.In Gaucher's disease, for example, the accumulation of glycocerebrosides in the lysosomes of cells of the mononuclear phagocytic system leads to the formation of lipid-loaded cells known as Gaucher cells.This accumulation of glycocerebrosides in spleen macrophages and liver Kupffer cells causes enlargement of these organs.In addition, other sphingolipidoses such as Niemann-Pick's disease, gangliosidoses and Fabry disease also present spleen manifestations 10 .The spleen participates in the metabolism of all metals, of albuminoids and of indirect bilirubin from phagocytized red blood cells 11,12 .
Patients with myeloproliferative diseases such as polycythemia vera and myelofibrosis usually course with reduced total cholesterol, HDL-cholesterol, apolipoprotein B and apolipoprotein A-1 levels.In these diseases, splenectomy normalizes the values of these substances 2,7,13 .
There is a clear and confirmed relationship between dyslipidemia and vascular diseases, especially atherosclerosis, which is responsible for the elevated morbidity and mortality of the general population.Fatouros et al. 2 observed that persons submitted to splenectomy due to trauma presented a higher incidence of coronary artery disease probably due to lipid disorders.The changes in lipid metabolism caused by splenectomy due to trauma may eventually explain the high incidence of acute myocardial infarction detected in World War II veterans 14 .Some experimental studies have reported the influence of the spleen on lipid metabolism.King 13 in 1914, detected an increase in cholesterol in dogs after spleen removal.Asai et al. 4 observed that rabbits fed products containing high cholesterol levels showed an increase in cholesterol, triglycerides and phospholipids, and low HDL-cholesterol levels after removal of the spleen.This fact was also reported in rats fed a cholesterol-enriched diet 2 .
Despite the extensive documentation about the dyslipidemias available in the literature, few and controversial studies have been reported about the correlation between the spleen and the effect of conservative splenic operations on lipid metabolism and about the efficacy of autogenous splenic tissue implants regarding the restoration of the metabolic function of the spleen.In view of this gap in the literature, the objective of the present study was to assess the influence of the spleen and of different operations performed on it and of a spleen auto-implant on the lipidogram.

Methods
The present study was conducted according to the recommendations of the International Norms for Animal Protection and of the Brazilian Code of Animal Experimentation (1988)   and was approved by the Ethics Committee of the Department of Surgery, Medicine School of the Federal University of Minas Gerais 16,17 .
Fifty female Wistar rats were housed in appropriate cages, up to five animals to a cage, at ambient temperature of 25°C and on a 12 hour light/12 hour dark photoperiod, with free access to water.They were fed with regular rodent chow during the pre and postoperative time of the experiment.Before the experiment, the rats were submitted to evaluation to rule out possible illness and were allowed to adapt for a period of 15 days.The animals were divided at random into the following groups: Group 1 (n=10) -Control: no surgical intervention.The weight of the not operated rats (Group 1), were also assessed on the same day.The surgical procedures were carried out under conditions of asepsis and antisepsis and under general anesthesia with intraperitoneal association of ketamine (10mg/kg) and xylazine (90mg/kg).The median laparotomy was closed with continuous sutures in two layers using 5-0 cotton sutures.
The animals were monitored daily for 120 days.Same amount of food and water was offered to all groups.At the end of the experiment, the rats were anesthetized with the same drugs used for the first operation.The body weight of all animals was assessed once more on this day.Through a median laparotomy, the caudal vena cava was identified and punctured for vacuum collection of 5ml of blood into tubes containing gel and covered with aluminum foil for protection from light.The tubes were then immediately submitted to laboratory analysis.Serum

Results
No adverse events were observed in experiment.The animals recovered rapidly from the surgical procedure during the immediate postoperative period.No difference in body weight was verified between the groups at the beginning or end of the experiment (Table 1).The HDL-cholesterol fraction of the group submitted to total splenectomy (Group 3) was lower than that recorded for the remaining groups (p=0.0026,ANOVA).There was no significant difference among other groups (Table 2).
The triglyceride determinations also presented values distributed within the normal curve (p>0.10)(KS distance of 0.2370 for Group 1, 0.1413 for Group 2, 0.2352 for Group 3, 0.1776 for Group 4, and 0.2559 for Group 5).The variances did not differ (p=0.2486 by the Bartlett test).There was no difference among the groups studied (p=0.1571,ANOVA) (Table 2).

Discussion
Particular emphasis is being currently placed on the spleen in medical practice because of its attributions, partially well known, such as its immune, filtering and hematopoietic roles 18 .
The respect due to this organ because of its importance in the physiopathology of many diseases has favored the development of multiple operations aiming at the preservation of the spleen, with a consequent reduction of sepsis rates and of postoperative hematologic disorders 19 .Total splenectomy, which used to be extensively indicated in the past, is being progressively replaced with spleen-conserving procedures such as spleen suture, partial splenectomy, subtotal splenectomy or autogenous spleen tissue implant after spleen removal 18,20,21 .More recently, the non-surgical approach to spleen trauma started to be well accepted, with better results than those obtained by surgery 22-24.Some clinical and experimental studies have evaluated the possible relation between the spleen and lipid metabolism in two antagonistic situations, i.e., hypersplenism [1][2][3][6][7][8] and after asplenia 1,2,5,7,9 . Gilert et al. 8 noted a reduction of serum levels of total cholesterol and its LDL and HDL fractions in patients with myeloproliferative diseases associated with hypersplenism.
Cholesterol levels were even more reduced during periods of worsening of the base disease.After total splenectomy or when the disease was controlled with chemotherapeutic agents, hypocholesterolemia was reversed 8 .Other authors also analyzed this relation in patients with type 1 Gaucher's disease 6 and type B thalassemia major 3 and obtained similar results.
On the other hand, in experimental studies, total splenectomy was accompanied by elevated cholesterol and triglyceride levels 2,4,5 .Aviram et al. 7 noted that the LDL fraction of cholesterol was elevated after splenectomy carried out in order to treat myeloproliferative disorders.
According to Fatouros et al. 2 cholesterol levels does not change after splenectomy.However the level of triglyceride increases in asplenic rats.All this findings are different from our results.These differences may be related to the species of the rats or to their gender.Even without literature support to any hypothesis related to these aspects, we do not believe these findings are related to the period of follow-up.On the order hand, we did not find any data suggesting that regular rodent show, or the weight and age of the animals may influence the lipidogram findings after splenectomy.
Dyslipidemia secondary to total splenectomy may result in a higher incidence of atherosclerotic disorders 25 .In an attempt to establish this relation, Asai et al. 5 observed that rabbits submitted to total splenectomy and receiving a cholesterol-rich diet presented atherosclerotic plaques in the aorta after four months.
Some theories have been proposed to explain the possible mechanisms implicated in the regulation of plasma lipids by the spleen 1,3,5-7 .Schmidt et al. 1 compared the spleen to a lipid reservoir that increases in situations of hypersplenism.By an increase in phagocytosis, spleen macrophages may accumulate large quantities of fat, with consequent hypolipidemia 1 .Another explanation for lipid reduction could be the autoimmune effect of the mononuclear phagocytic system against the structures found in the HDL-cholesterol and LDL-cholesterol lipoproteins, resulting in their plasma clearance 3,6 .
On the basis of these theories, after total splenectomy the inverse effect may provoke an increase in the serum levels of plasma lipids.According to Asai et al. 5 , a "splenic factor" is related to hypocholesterolemia by decreasing the serum cholesterol level.
Thus in an asplenic condition, this factor is missed and metabolism reactions enhance serum cholesterol levels.However, there is no evidence of lipid metabolism in spleen cells, even knowing the spleen storage of phospholipids in dislipidemias such as Gaucher´s disease 1,2,4,6,13,25 Further investigation of this phenomena is needed to understand the relation between spleen and lipid metabolism.

Group 2 (
n=10) -Sham-operation: animals submitted to laparotomy and laparorrhaphy.Group 3 (n=10) -Total splenectomy: ligature of the spleen vascular pedicle performed with 5-0 chromic catgut and the spleen was completely removed.Group 4 (n=10) -Subtotal splenectomy: ligature of the vascular pedicle of the inferior pole of the spleen with 5-0 chromic catgut preserving the splenogastric vessels, and section of this segment, followed by running suture of the remnant upper pole with 5-0 chromic catgut.Group 5 (n=10) -Total splenectomy complemented with an autogenous spleen tissue implant in the omentum: ligature of the spleen vascular pedicle with 5-0 chromic catgut and full extraction of the organ.The spleen was sliced in 5 segments which were sutured to the anterior surface of the omentum with 5-0 chromic catgut.The body weight of the animals of groups 2, 3, 4 and 5 was verified immediately before the surgical procedure.The day of the operation was considered as the first day of the experiment.
concentrations of triglycerides, total cholesterol, very low density lipoprotein (VLDL-cholesterol), high density lipoprotein (HDLcholesterol) were determined by the enzymatic colorimetric method, after centrifugation at 4000 rpm for 10 minutes.The low density lipoprotein (LDL) was measured by mean of the Friedewald formula: LDL-cholesterol = total cholesterol-HDLcholesterol -(triglycerides/5).Then the animals were killed with an excess dose of intraperitoneal association of ketamine and xylazine.Data concerning about the body weight of the animals and the serum total cholesterol and its fractions and triglycerides were analyzed statistically in each group by the Kolmogorov-Smirnov (KS) test to determine Gaussian distribution of the data, and then by the Bartlett test to determine the variances.When the data presented Gaussian distribution and the same variance, they were submitted to analysis of variance (one-way ANOVA) followed by the multiple comparison Tukey-Kramer test.The level of significance was set at p<0.05 in all analyses.

TABLE 1 -
The body weight (g) of the animals assessed at the beginning of the experiment and four months after the spleen operations.Values are reported as means ± SD.

TABLE 2 -
Cholesterol fractions and triglycerides of control and rats submitted to spleen operations.Values are reported as means ± SD.
3: p = 0.0151 (Group 3 compared with the other groups)