Abstract
Background: Acromegalic patients have increased cardiometabolic risk factors due to an elevation of growth hormone (GH) levels. Human serum paraoxonase (PON), a high-density lipoprotein (HDL)-related enzyme, is one of the major bioscavengers and decreases the oxidation of low-density lipoprotein (LDL), a key regulator in the pathogenesis of atherosclerosis. In this study, we investigated a potential relationship between serum PON levels or PON polymorphisms and acromegaly.
Methods: A total of 48 acromegalic patients and 44 healthy controls were included in this study. Serum GH levels, insulin-like growth factor-1 levels and lipid profiles were measured. Serum PON levels, as well as PON 1 L55M and Q192R gene polymorphisms, were examined.
Results: No significant differences were found in terms of age, gender, presence of diabetes, serum LDL cholesterol (LDL-C), HDL-C, or triglyceride levels between the case and control groups (P > 0.05). A statistically significant difference was found in serum PON levels between the cases and controls (P = 0.007). The median serum PON level was 101 ± 63.36 U/l in the case group and 63 ± 60.50 U/l in the control group. There was a significant correlation between serum PON levels and IGF-1 levels (P = 0.004, r = 0.319); however, no significant differences were found in PON1 L55M and PON Q192R polymorphisms between the patients and controls (P = 0.607 and P = 0.308, respectively). In addition, no significant differences were found in serum PON levels in acromegalic patients who were and were not in remission (P = 0.385), nor between those with PON1 L55M and Q192R polymorphisms (P = 0.161 and P = 0.336, respectively).
Conclusions: Elevated serum PON levels were detected in acromegalic patients, independently of their remission status. This suggests protective effects for cardiometabolic risk parameters.
Keywords:
Introduction
Acromegaly is a rare endocrine disease that results from the excess production of growth hormone (GH) from the pituitary gland.Citation1 It is associated with metabolic diseases (increased hypercoagulability, insulin resistance, diabetes mellitus, hypertension, and hyperlipidemia) that cause an increased risk of atherosclerosis.Citation2 Few studies have shown an association between acromegaly and coronary artery diseases, with conflicting results.Citation2–Citation8 For example, some studies have reported an increase in atherosclerotic diseases in acromegaly, whereas others have reported a decreased risk.Citation2–Citation4,Citation7,Citation8
Oxidative stress caused by reactive oxygen species (ROS) triggers endothelial dysfunction and atherosclerosis.Citation9 Oxidized low-density lipoprotein (LDL), a proatherogenic molecule, is formed by the effects of ROS on LDL.Citation10 Some conflicting results regarding an association between GH, IGF-1, and oxidative stress have been reported. In some studies, increased levels of oxidative stress resulted in a reduction in antioxidant capacity,Citation11–Citation14 whereas in others, no increase in oxidative stress levels was found; moreover, a reduction in lipid peroxidation was reported.Citation15–Citation19
Human serum paraoxonase (PON) is an enzyme related to high-density lipoprotein (HDL), and hydrolyzes aromatic carboxylic acid esters, organophosphates, and carbamates.Citation20 PON F is under genetic control and variants of the PON1 gene strongly influence PON activity.Citation21 The PON1 gene has been mapped to chromosome 7q21–22.Citation22 The most common polymorphisms within the PON1 gene are the glutamine/arginine substitution (Q/R) at codon 192 and the leucine/methionine substitution (L/M) at codon 55.Citation23 PON plays a role in inflammation and lipid metabolism, and decreases the oxidation of LDL, a key regulator in the pathogenesis of atherosclerosis. To date, there is no information regarding the clinical significance of the relationship between serum PON levels or PON polymorphisms and acromegaly.
Materials and methods
Subjects
A total of 48 patients diagnosed with acromegaly (23 patients in remission and 25 not in remission) and 44 age- and gender-matched controls were included in this study. Informed consent was obtained from all of the participants, and the study was approved by the local Ethics Committee of Bulent Ecevit University, Faculty of Medicine (Zonguldak, Turkey). Background medical histories of all of the patients were evaluated retrospectively. Patients were included in this study as they presented typical clinical features of acromegaly and increased GH and IGF-1 levels compared to corresponding gender- and age-matched participants. Acromegaly diagnosis was confirmed when GH >1 µ/l was reached following a suppression test with 75 g oral glucose and high IGF-1 levels compared to age- and gender-matched subjects. Remission criteria were defined as GH <1 ng/ml following suppression of 75 g glucose and a normal range of IGF-1 levels according to age and sex. Both patients and controls had normal renal and hepatic functions without cardiovascular or cerebrovascular disease, peripheral vascular disease, acute or chronic infection, uncontrolled hormonal deficiency, hormonal co-production, were not younger than 18 years of age, and were not being treated with antioxidants or antihyperlipidemic therapy.
Laboratory measurements
Blood samples were drawn from the ante-cubital vein into closed tubes the morning after 12 hours of fasting and were sent to the laboratory within 1 hour. Serum levels of glucose, total cholesterol, triglycerides, and HDL-C were determined using a BS-2000M autoanalyzer (Mindray, Shenzhen, China). LDL-C was calculated using the Friedewald equation: LDL-C = total cholesterol – (HDL-C + triglycerides/5).Citation24 Serum GH and IGF-I levels were measured using a solid-phase, enzyme-labeled chemiluminescent immunometric assay (CLIA) with an Immulite 2000 analyzer supplied by Siemens Medical Solutions Diagnostics (Los Angeles, CA, USA).
Serum PON measurement
Blood samples were centrifuged and aliquots immediately stored at −80°C until use. Serum PON levels were measured using a Shimadzu UV 1601 spectrophotometer (Shimadzu Co., Kyoto, Japan) according to the definition of Gan et al.Citation25 with an intra-assay CV <8.8%. Paraoxon (1 mM) was used as the substrate in the presence of 1 mM CaCl2 in 100 mM Tris–HCl buffer (pH 8.0); serum was added to start the reaction, and the increase in absorbance at 412 nm was recorded. The amount of p-nitrophenol was calculated from the molar extinction coefficient (17.100 M−1 cm−1). Results are expressed as U/l.
PON polymorphism (DNA analysis)
Genomic DNA was extracted from peripheral blood leucocytes (200 µl total blood) using a Macherey-Napel (MN) Nucleospin blood® DNA Extraction Kit (cat no. 740.951.250), according to the manufacturer's instructions. A PCR-based restriction fragment length polymorphism (RFLP) method was used to detect PON1 L55M and Q192R gene polymorphisms. Amplifications were performed in 0.2 ml thin-wall tubes of 50 µl aliquots containing 50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, 0.5 µM each of the four deoxynucleotides, 50 pmol each primer, 1 U Taq DNA polymerase, and 5 µl the extracted DNA. The primary sequences for amplifying each single nucleotide polymorphism (SNP) are shown in Table . PCR products (15 μl) were digested overnight with the appropriate restriction enzyme (Table ), and digests were analyzed by electrophoresis on a 3% agarose gel.
Table 1 The primary sequences for amplifying each SNP for PON polymorphisms.
Statistical analysis
Statistical analyses were performed with SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Distribution of data was determined by Shapiro–Wilks test. Continuous variables are expressed as the mean ± standard deviation or median (minimum–maximum), and categorical variables were expressed as frequency and percent. Continuous variables were compared to the independent samples t-test or Mann–Whitney U test, and categorical variables were compared using Pearson Chi-square test. A P value less than 0.05 was considered statistically significant.
Results
The study included 48 acromegaly patients and 44 controls. The median age of patients was 48.0 years (range, 21–73 years), and the median age of controls was 40 years (range, 20–66 years) (P = 0.058). A total of 27 (56.2%) patients and 33 (75%) controls were female (P = 0.114). Disease duration ranged from 1 to 6 years. The groups of acromegaly and controls were similar in terms of the presence of diabetes (P = 0.177). Although there was a significant difference in IGF-1 levels (P ≤ 0.01), there were no significant differences in serum LDL-C, HDL-C, or triglyceride (TG) levels between the case and control groups (P > 0.05). A statistically significant difference was also found in serum PON levels between cases and controls (P = 0.007). The median serum PON level was 101 ± 63.36 U/l in the case group and 63 ± 60.50 U/l in the control group (Table ). The LL polymorphism was associated with increased serum PON levels. No differences in the Q192R polymorphism were observed (Table ).
Table 2 Demographical and laboratory parameters of patients and controls.
Table 3 Serum paraoxonase level in each polymorphism.
No significant difference in the distribution of PON1 L55M polymorphisms was found between cases and controls (P = 0.607). The frequencies of the PON1 L55M polymorphisms were 58% (LL), 35% (LM), and 6% (MM) in the case group and 64% (LL), 34% (LM), and 2% (MM) in the control group. The distribution of PON1 Q192R polymorphisms was similar between the case and control groups (P = 0.308). The frequencies of the PON1 Q192R polymorphisms were as follows: QQ (n = 36, 75%), QR (n = 11, 23%), and RR (n = 1, 2%) in the case group and QQ (n = 38, 86%), QR (n = 6, 14%), and RR (n = 4, 0%) in the control group (Table ).
Table 4 PON polymorphisms of patients and controls.
No significant difference was found in serum PON levels between acromegalic patients who were in remission and those who were not in remission (P = 0.385), nor between those with PON1 L55M and Q192R polymorphisms (P = 0.161 and P = 0.336, respectively) (Table ). Although there was a significant correlation between serum PON levels and IGF-1 levels (P = 0.004, r = 0.319), there was no correlation between serum PON levels and lipid panels (P > 0.05) (Table ).
Table 5 PON polymorphisms of acromegalic patients with or without remission.
Table 6 Correlation of serum of PON-1 level with IGF-1 and lipids.
Discussion
We found that serum PON levels were significantly higher in acromegalic patients than in gender- and age-matched controls. In addition, we found a correlation between serum PON levels and IGF-1 levels, but did not observe any correlation between serum PON levels and lipid levels. There were no differences in terms of allelic frequencies between the patients and controls, and these polymorphisms did not affect serum PON levels.
Despite an increased frequency of diabetes, hypertension, and hyperlipidemia in acromegalic patients, studies that have investigated a potential association between acromegaly and atherosclerosis are debatable, and there is no consensus regarding coronary artery disease in acromegalic patients, as mentioned above.Citation2–Citation8 Studies investigating the association between oxidized LDL and acromegaly also have conflicting results. Yarman et al.Citation11 suggested that an increase in GH levels can directly affect lipid peroxidation. Other studies also supported this study and found that anti-oxidative enzyme levels were decreased.Citation12–Citation14
Ozkan et al.Citation16 found that oxidized LDL levels were lower in the active acromegaly group than in the controlled acromegaly group, although the difference was not significant. This group also found that high mobility group box 1 protein (HMGB1) and total anti-oxidative capacity, indicators of oxidative injury, and lipid panels were similar in both the active and controlled acromegaly groups. C-reactive protein was lower in the active acromegaly group. The authors even suggested that oxidative stress and inflammation do not contribute to the atherosclerotic process in acromegalic patients.
Sukhanov et al.Citation17 showed that IGF-1 has anti-atherogenic effects by decreasing lipid peroxidation and formation of foam cells. IGF-1 also has a role in atherosclerotic plaque stabilization. Although some studies have reported the anti-atherogenic effects of GH and IGF-1, it is suggested that IGF-1 increases the release of endothelial nitric oxide, which regulates vascular blood flow.Citation5,Citation15 It has also been reported that IGF-1 exhibits anti-inflammatory effects.Citation18
PON1, an HDL-associated hydrolytic and antioxidant 355 amino acid glycoprotein enzyme that is synthesized in the liver and secreted into the blood, tends to restore redox balance, thus protecting vessels from atherosclerotic progression.Citation25,Citation26 The metabolic activity of HDL can prevent the oxidation of LDL, which might be mediated by certain enzymes.Citation27 It has been shown in vitro that PON reduces the accumulation of lipid peroxidation products on LDL.Citation28 LDL oxidation may be induced under conditions of oxidative stress, which is physiologically counteracted by hydrosoluble, liposoluble, and enzymatic (one of them being PON1) antioxidants.Citation29 To the best of our knowledge, this is the first report to investigate serum PON activity and PON polymorphisms in acromegalic patients.
An association between PON gene polymorphisms and coronary heart disease has been reported in some studies,Citation30,Citation31 whereas no association has been reported in others.Citation32,Citation33 In this study, we found no statistically significant difference in PON Q192R or L55M polymorphisms between the cases and controls (P = 0.334). Serum PON levels are reduced in patients with coronary artery disease.Citation34 Boero et al.Citation35 did not find any statistical difference in serum PON levels between acromegalic patients and controls. In contrast, we found that serum PON level was higher in patients with acromegaly than the controls (P = 0.007). Similar to our results, Liberopoulos et al.Citation36 reported an elevation of serum PON levels in patients undergoing peritoneal dialysis.
Previous studies have noted an increase in PON activity for both RR and LL genotypes.Citation34,Citation36–Citation40 However, in our study, serum PON levels were not affected by PON L55M or PON Q192R (P = 0.480), nor by the subtypes of these gene polymorphisms. In another study, the PON1 Q192R polymorphism was a risk marker for insulin resistance, a pathological factor involved in the development of metabolic syndrome.Citation41 In addition, we did not find any statistically significant difference in serum lipid parameters between RR and LL genotypes.
Some studies have reported a decline in PON activity of diabetic patients.Citation39,Citation40 In our study, we did not find any significant difference in serum PON levels when we compared diabetic and non-diabetic patients (P = 0.393). To date, there are no reports in the literature that have investigated serum PON levels and PON polymorphisms in acromegalic patients. Here, we found an increase in the PON activity of acromegalic patients, which was positively correlated with IGF-1 levels.
The limitations of this study included its small number of acromegaly patients. There was also no evaluation of the association between cardiovascular disease and PON activity in acromegalic patients. The disease duration of our patients was short (<6 years), and the median age of our patients was young. The reason for increased PON activity was hypothesized to be because the main source of paraoxonase is the liver, and GH likely induces the production of nonspecific proteins like paraoxonase.
In conclusion, our study indicates that increased serum PON level, likely induced by increased serum IGF-1 levels, has a protective effect, as our acromegalic patients exhibited a lower risk for cardiometabolic situations.
Disclaimer statements
Contributors All authors contributed equally.
Funding None.
Conflict of interest All authors declare that no conflict of interest.
Ethical approval Paper has received ethical approval.
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