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It has been suggested that plant sterol absorption is increased in type 1 diabetes mellitus (T1DM) and that this may relate to the increased cardiovascular risk seen in T1DM. The cardiovascular benefit of lowering low-density lipoprotein–cholesterol with statin medication has also been shown to be influenced by plant sterol absorption.
The relationship between sterol concentrations, coronary artery disease (CAD), and the use of statin medications in T1DM was compared between participants with CAD (Minnesota codes 1.1, 1.2, 1.3, 4.1–4.3, 5.1–5.3, and 7.1; n = 82), from the Pittsburgh Epidemiology of Diabetes Complications (EDC) study, and those without (n = 213). Serum sterol concentrations reflecting cholesterol absorption (β-sitosterol and campesterol) and synthesis (desmosterol and lathosterol) were assayed and analyzed by gas chromatography and were expressed as a ratio of total cholesterol (×103).
No differences were observed in markers of cholesterol absorption between individuals with and without CAD. In patients with CAD, significantly lower levels were observed for both sterol markers reflecting cholesterol synthesis compared with individuals without CAD [desmosterol: 0.34 vs 0.42, respectively (P = 0.003); lathosterol 0.47 vs 0.54, respectively (P = 0.019)]. Further stratification by statin medication use revealed significantly lower levels of synthesis-reflecting sterols in individuals taking statin medication, particularly those with CAD.
Although previous reports suggest that higher levels of cholesterol absorption in T1DM potentially increase cardiovascular risk in this population, the present data suggest no differences in cholesterol absorption between T1DM individuals with and without CAD.
Plant sterols have been associated with coronary artery disease (CAD), such as in the genetic condition of sitosterolemia, in which 20-fold increases in plant sterol levels are related to early and excessive CAD and aortic valve disease.1 Furthermore, sterols have been identified in atherosclerotic lesions from individuals with apparently normal cholesterol.2 Previous data suggest that type 1 diabetes mellitus (T1DM) may be associated with low cholesterol synthesis and high cholesterol/plant sterol absorption,3–5 findings that appear to contrast with those seen in type 2 diabetes, in which the opposite may occur.6 Thus, if increased plant sterol concentrations occur in T1DM as a result of increased absorption, plant sterols may contribute directly to the excess development of CAD seen in this population.
Non-cholesterol sterols circulating in human sera have been reported to be reliable markers of cholesterol metabolism, reflecting both cholesterol absorption and synthesis.5 The synthesis of cholesterol has been shown to be positively associated with sterol synthesis intermediates (e.g. desmosterol and lathosterol) and negatively associated with sterols related to absorption (e.g. campesterol and β-sitosterol).5 In addition to their association with cholesterol metabolism, plant sterols are known to compete with dietary and biliary cholesterol for intestinal absorption, which is further associated with reductions in serum cholesterol concentrations.7 Such associations have led to the identification of plant sterols as novel anti-atherosclerotic risk factors and their use as a cholesterol-lowering therapy. Despite plant sterols being widely marketed for their cardiovascular benefit in a wide variety of foods, beverages, and dietary supplements, questions regarding the safety of these compounds have been circulating for many years.8
The effective lowering of low-density lipoprotein–cholesterol (LDL-C) by the use of statins is widely reported to reduce CAD, including CAD mortality.9,10 The CAD benefit of lowering LDL-C with statin use has also been shown to be influenced by sterol synthesis and absorption. High absorbers may be less likely to respond to statin therapy, as suggested by the analysis of the 4S study,11 in which a 2.2-fold difference was noted in the recurrence of CAD events in those treated with simvastatin according to their quartile of absorption. Of particular relevance to this proposal is the more recent observation that T1DM subjects appear to be, in general, high absorbers and low synthesizers4 and, thus, may be less responsive to statin therapy. Therefore, in the present study, we examined the relationship between plant sterol markers of cholesterol absorption and synthesis, the use of statin medication, and CAD status in participants from the Pittsburgh Epidemiology of Diabetes Complications (EDC) study, a prospective study of childhood-onset T1DM currently in its 20th year of data collection.
The subjects of the present investigation were participants in the Pittsburgh EDC study, a 20-year prospective follow-up study of childhood-onset T1DM that has been described in detail elsewhere.12 Briefly, participants were diagnosed (or seen within 1 year of diagnosis) between 1950 and 1980, before 17 years of age, at the Children’s Hospital of Pittsburgh. This population has been shown to be representative of the T1DM population in Allegheny County, Pennsylvania.13 Analyses for the current cross-sectional investigation were performed using data collected from the 18-year examination cycle (2004–2007; n = 295).
As part of the EDC examination, height was measured using a stadiometer and weight was measured on a Detecto (Cardinal Scale Manufacturing, Webb City, MO, USA) physician scale. Standardized sitting blood pressure and heart rate were measured after a 5-min rest period.14 Fasting blood samples were analyzed for sterol markers associated with cholesterol synthesis (desmosterol and lathosterol) and absorption (campesterol and β-sitosterol). Total cholesterol was measured enzymatically.15 High-density lipoprotein–cholesterol (HDL-C) levels were determined by a precipitation technique (heparin and manganese chloride) with modification of the Lipid Research Clinics method.16 Non-HDL-C levels were calculated by subtracting HDL-C from total cholesterol. Blood samples were analyzed for hemoglobin A1c (HbA1c) using the DCA 2000 analyzer (Bayer Diagnostics, Tarrytown, NY, USA).
Waist and hip circumferences were assessed and converted to a waist:hip ratio (WHR) as a measure of visceral adiposity. Estimated glucose disposal rate (eGDR), a measure of insulin sensitivity, was calculated using a regression equation (involving HbA1c, WHR, and hypertension status) derived from hyperinsulinemic–euglycemic clamp studies.17 Results from at least two of three timed urine collections (24-h, overnight, and random-timed post-clinic visit urine) were used to determine albumin excretion rates (AER). Both a standardized medical history and a clinical examination were performed by a trained internist to classify participants according to CAD status. In the present study, CAD comprised a positive clinical history [EDC clinic physician-diagnosed angina, myocardial infarction (either pathological Q waves (Minnesota codes 1.1, 1.2) or ischemic ECG (Minnesota codes 1.3, 4.1–4.3, 5.1–5.3, 7.1)) at the time of examination or on review of previous hospital records, or angiographic evidence of ≥50% stenosis with or without revascularization]. The use of lipid-lowering medication was assessed by participant responses on the EDC medical history questionnaire. All current medications, along with the medication dose and reason for taking the medication, were recorded. All participants taking 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins or statin combination drugs), ezetimibe, probucol, dexthrothyroxine, nicotinic acid and derivatives, and bile acid sequestrants were classified as taking LDL-C-lowering medication. Individuals taking an LDL-C-lowering medication who were not taking any form of statin were excluded from analyses (i.e. ezetimibe, probucol, dexthrothyroxine, nicotinic acid or derivatives, or bile acid sequestrants alone; n = 7). The study protocol was approved by the University of Pittsburgh Institutional Review Board.
Fasting serum samples were analyzed for sterol concentrations by capillary gas chromatography (GC) after saponification, extraction, and derivatization. Briefly, samples were mixed with methanolic 3 mol/L NaOH and then heated at 90°C for 1 h, cooled, and extracted with petroleum ether. The combined extracts were dried under nitrogen and 20 μL bis(trimethylsilyl)trifluoroacetamide (BSTFA; Supelco, Bellefonte, PA, USA) was added. Samples were then heated at 75°C for 15 min and, after cooling, 1 μL was injected onto a GC column (Equity-1; 100 m × 0.32 mm × 1.0 μm; Supelco). The gas chromatograph used was a Perkin-Elmer (Boston, MA, USA) 8420 equipped with a flame ionization detector. The oven, injector, and detector temperatures were 275, 290, and 290°C, respectively. Helium was the carrier gas at 103.43 kPa. Identification of components was by comparison with those of authentic standards (Steraloids, Newport, RI, USA). Conversion factors were determined using known amounts of authentic standards. The integrator used was a Shimadzu C-R3A chromatopac (Shimadzu, Kyoto, Japan). All assays were performed in the Heinz Nutrition Laboratory, Department of Epidemiology, University of Pittsburgh Graduate School of Public Health. Interassay coefficients of variation ranged from 0.95% (β-sitosterol) to 6.70% (campesterol).
Plant sterol values are expressed as a ratio of total cholesterol (×103). Non-parametric variables (e.g. campesterol, β-sitosterol, desmosterol, lathosterol, diabetes duration, body mass index (BMI), HbA1c, total cholesterol, HDL-C, and triglycerides) were transformed by natural log. Non-parametric variables that could not be normalized by natural log transformation (AER) were analyzed with non-parametric tests. Group differences were examined by Student’s t-test and Mann–Whitney U-test, as appropriate. Because unequal numbers of men and women were observed between groups when stratifying according to statin medication use, comparisons between CAD status and statin medication use were adjusted for gender. General linear models were used to obtain P values for differences between groups adjusting for gender. P<0.05 was considered significant. Analyses were performed using spss for Windows software v. 15 (SPSS, Chicago, IL, USA)
The characteristics of the 295 study participants with T1DM are presented according to CAD status in Table 1. Participants with CAD were older, had had diabetes for a longer duration, and had higher levels of albumin excretion. After adjusting for gender, individuals with CAD were also found to have higher WHR, systolic blood pressure (SBP), and triglyceride levels, and a lower heart rate and total cholesterol and LDL-C levels. A significantly lower eGDR rate also suggested higher insulin resistance in those subjects with CAD. There was no significant difference in HbA1c, HDL-C, non-HDL-C, and BMI between the CAD groups.
Across all participants, a positive correlation was observed between values of plant sterols reflecting cholesterol absorption (campesterol and β-sitosterol; r = 0.92, P < 0.001); a similar positive association was observed between values of sterols reflecting cholesterol synthesis (lathosterol and desmosterol; r = 0.65, P < 0.001). Significant inverse correlations between the plant sterols reflecting cholesterol absorption and synthesis were also observed consistently (Table 2).
The values of plant sterols reflecting cholesterol absorption (campesterol and β-sitosterol) were not significantly different between individuals with and without CAD (Table 3), yet values of plant sterols reflecting cholesterol absorption were noted to be lower in subjects with CAD (Table 3). The levels of both sterols reflecting cholesterol synthesis (desmosterol and lathosterol) were significantly lower in T1DM individuals with CAD. Further stratification by statin medication use (Table 4) revealed no differences in plant sterols reflecting cholesterol absorption, regardless of CAD status. However, values of sterols reflecting cholesterol synthesis were significantly lower in individuals taking statin medication, with the lowest levels observed in subjects with CAD taking statin medication. No significant interactions between gender and CAD status and gender and statin medication use were observed for any of the sterol markers. Among those subjects with CAD, further categorization by soft (reported angina or ischemic ECG) and hard (myocardial infarction) CAD did not alter the findings, although significance was reduced.
The data of the present study do not support the hypothesis that increased plant sterol absorption is related to the presence of CAD in T1DM. Although not significant, the results from the present investigation may suggest lower levels of cholesterol absorption in T1DM individuals without CAD compared with those with CAD. Although a previous report from Miettenen et al.4 showed significantly higher levels of cholesterol absorption sterols in T1DM compared with a similar type 2 diabetic population, the present study examined only T1DM individuals with and without CAD. Therefore, the present findings suggesting no differences in cholesterol absorption in T1DM individuals with and without CAD cannot be used to answer the question of whether an overall difference in the level of cholesterol absorption exists in T1DM compared with the general population.
Even though no significant associations were observed in markers of cholesterol absorption, lower levels of cholesterol synthesis sterols were seen in T1DM individuals with CAD. The use of LDL-C-lowering medications, such as statins, which decrease cholesterol synthesis, was found in 43.2% of participants at the 18-year examination and was associated with lower markers of cholesterol synthesis. Consequently, the overall lowest levels of synthesis sterols were observed in subjects with CAD who were using statin medication, a notable finding that is worthy of further follow up. It is particularly striking that no difference in synthesis sterols was observed between CAD groups in individuals not taking statin medication, because it may have been expected that synthesis would be elevated in untreated individuals, albeit lower in subjects with CAD, secondary to treatment. The lower levels of synthesis markers among individuals with CAD using statin medication may have reflected a greater premorbid tendency towards cholesterol synthesis in subjects with CAD who may have subsequently experienced an increased response to statin therapy. In addition, no indication of increased sterol synthesis was seen in subjects with CAD not taking statin medication, although the number of such cases was smaller. Although, only a small number of EDC participants actually started using statin medication prior to the presence of clinical CAD (n = 8), it remains possible that most of the individuals with T1DM and CAD were not taking lipid-lowering therapy at the time of CAD and that they may have had high baseline levels of synthesis sterols prior to presentation of clinical disease. It is therefore more likely that lipid-lowering therapy was initiated at the time of diagnosis of CAD and the present data most likely reflect lower cholesterol synthesis in these subjects as a result of the therapeutic use of lipid-lowering medications. However, it remains unclear whether T1DM patients with elevated cholesterol synthesis, identified by serum synthesis sterols, are at increased risk of CAD and whether the prophylactic use of LDL-C-lowering medication may decrease the risk of CAD.
Some strengths and limitations of the present study should be noted. The T1DM participants in the EDC study are a representative sample of T1DM individuals with a long duration of the disease. With this strength comes an inherent weakness in that this sample includes individuals with macro- and microvascular complications, such as nephropathy, which influences dyslipidemia. Because individuals with CAD had higher AER, the potential influence of this renal complication on cholesterol metabolism cannot be ignored. Further EDC analyses revealed no significant associations between AER, estimated glomerular filtration rates, or albumin/creatinine ratios with sterol levels (data not shown), suggesting a limited influence of renal function on sterol levels in this sample of T1DM patients. In addition, the ability to investigate the effect of statin medication on sterol levels between CAD groups is an important strength of the study because the reasons for the reported alterations in sterols during statin treatment are unknown and require further investigation. Finally, the sample size of the present study is much larger compared with similar investigations exploring sterol levels in T1DM,3,4,18–20 which may add further statistical power to our results. A limitation of the present study is that dietary records were not collected from the participants and the effect of recent dietary habits on circulating sterol levels cannot be accounted for. Additional studies on this topic are clearly warranted. Future investigations should examine whether sterol concentrations are associated with a risk for the development of CAD and whether reductions in LDL-C by additional lipid-lowering agents, such as ezetimibe, produce greater than expected reductions in CAD risk for the reductions in cholesterol; however, given recent results,21 this seems unlikely. The cross-sectional nature of the present study only allows the examination of associations between plant sterol levels and the presence of CAD in T1DM; further longitudinal investigations are required to elucidate the causal/temporal relationship between the use of statin medication and its effect on the risk of the development of CAD as it may be reflected by circulating sterol levels in T1DM populations.
In conclusion, in our subjects with T1DM, plant sterol markers associated with cholesterol absorption are not increased in individuals with CAD. However, sterol markers of cholesterol synthesis in those using LDL-C-lowering medications are lower, particularly in subjects with CAD.
This research was supported by National Institutes of Health Grants RO-1 DK071487–01 and RO-1 034818–23.
Disclosure None of the authors has a conflict of interest or any involvement, financial or otherwise, to disclose.