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Intra-individual variations of fasting plasma lipids, apolipoproteins and postprandial lipemia in familial combined hyperlipidemia compared to controls
Clinica Chimica Acta 328 (2003) 139 – 145 www.elsevier.com/locate/clinchim
Intra-individual variations of fasting plasma lipids, apolipoproteins and postprandial lipemia in familial combined hyperlipidemia compared to controls D. Delawi, S. Meijssen, M. Castro Cabezas * Department of Vascular Medicine F02.126, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands Received 10 July 2002; received in revised form 30 October 2002; accepted 1 November 2002
Abstract Background: The intra-individual variability of plasma lipids and apolipoproteins has not been studied systematically in familial combined hyperlipidemia (FCHL). Methods: Intra-individual changes in fasting plasma lipids and apolipoproteins B and AI were determined in 18 untreated FCHL subjects and 16 unrelated, normolipidemic subjects. Participants were matched for gender, age and body mass index. The mean follow-up period of fasting plasma lipids was 48.91 F 35.46 (mean F S.D.) days. Postprandial lipemia was determined on 3 different days in 1 week in 90 healthy controls and 17 untreated FCHL subjects by the area under the diurnal capillary triglyceride curve (TGc-AUC). Results: The coefficients of variation (CVs) for fasting plasma TG were similar between FCHL (23.2 F 10.2%) and controls (20.4 F 8.2%). The CVs for HDL-C, apo B and apo AI were the lowest of all fasting plasma measurements in both groups and there was no significant difference between FCHL (12.8 F 8.2%, 13.2 F 15.8% and 6.4 F 5.2%, respectively) and controls (11.4 F 4.3%, 11.3 F 10.6% and 7.8 F 4.6%, respectively). The CVs for postprandial lipemia were not different between FCHL (15.9 F 11.3%) and controls (15.1 F 11.0%), and were significantly lower than the CV of fasting capillary TG (TGc) in the same period (36.3 F 24.7% and 24.9 F 17.2%, respectively). Conclusions: Our study does not provide evidence for short-term major changes in fasting or postprandial lipemia or apolipoproteins in FCHL when systematically compared to healthy controls. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Triglycerides; Familial combined hyperlipidemia; Atherosclerosis; Postprandial lipemia; Apolipoproteins
1. Introduction Familial combined hyperlipidemia (FCHL) is the most frequent dominantly inherited disorder of lipid metabolism leading to increased risk for atherosclerosis [1 –7]. The diagnosis is based on clinical criteria * Corresponding author. Tel.: +31-30-2507399; fax: +31-302518328. E-mail address: [email protected] (M. Castro Cabezas).
such as the presence of ‘‘multiple-type hyperlipidemia’’ [1,3,5,6] in relatives of index patients, increased concentrations of plasma apolipoprotein (apo) B and a positive family history of premature cardiovascular disease (CVD). The genetic basis of FCHL has not been elucidated, although several groups have provided evidence suggesting that different genes are involved in the pathogenesis of this disorder [8 – 10]. Several metabolic characteristics of FCHL have been described during the last 20 years. Patients with
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0009-8981(02)00420-5
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FCHL are characterised by hepatic apo B overproduction [11 –13], delayed clearance of chylomicron remnants [14,15], insulin resistance [14,16,17], a primary defect in the metabolism of triglycerides and plasmafree fatty acids [14,18] with enhanced hepatic flux of free fatty acids resulting in exaggerated production of postprandial ketone bodies [19]. In addition, in a subset of patients, decreased activity of postheparin plasma lipoprotein lipase has been described [20,21]. One of the earliest described clinical characteristics was the presence of different lipoprotein phenotypes at one time point in members of a family unit [1,2,5]. This has been called ‘‘multiple-type hyperlipidemia’’. In addition, several investigators have observed that one individual patient may show different phenotypes during a longer period of time and have used the term ‘‘multiple-type hyperlipidemia’’ in this context. This was based on an early report by Brunzell et al. [22]. However, no systematic studies have been published on this subject and the metabolic basis for changing phenotypes in individual FCHL patients is not known. In non-FCHL subjects, fasting plasma lipids are widely variable [23,24]. The purpose of our study was to establish the variability of lipoprotein patterns in FCHL subjects on their regular diet. Since medication will affect the phenotypes, we evaluated the variation of the fasting lipoproteins of a group of FCHL index patients who were off medication for at least 1 month. In addition, the short-term variability of postprandial lipemia using diurnal capillary triglyceride profiles was evaluated and compared to healthy controls.
lipid lowering therapy and a positive family history of premature coronary heart disease (CHD) defined as myocardial infarction or cerebrovascular disease before the age of 60 in at least one blood-related subject of the index patient. In addition, the patients fulfilled the following inclusion criteria: absence of xanthomas, BMI < 30 kg/m2, absence of apo E2/E2 genotype, no use of drugs affecting lipid metabolism including lipid lowering drugs for at least 4 weeks before inclusion in this study and consumption of less than three units of alcohol/day. FCHL patients were on a low fat, low cholesterol diet. All FCHL patients participated in an ongoing study aimed to evaluate the effects of lipid lowering drugs on postprandial lipemia evaluated by oral fat loading tests. The results of the oral fat loading tests will be published separately. Normolipidemic, healthy controls (90) without a family history of cardiovascular disease or diabetes, absence of apo E2/E2 genotype and not using drugs known to affect lipid metabolism were recruited by advertisement. On the morning of inclusion, blood was collected after a 10-h fasting period for measurements of lipids, apolipoproteins, insulin and glucose. In addition, 16 different controls fulfilling the same inclusion criteria were selected to participate in the fat loading tests as matched healthy controls for the FCHL subjects. In order to be included, these controls needed to have the same anthropometric characteristics and age as the FCHL subjects. These 16 subjects visited our department on several occasions during a longer period of time and extensive fasting lipoprotein analysis was performed. 2.2. Capillary TG self-measurements
2. Subjects and methods 2.1. Subjects The study protocol was approved by the Human Investigations Review Committee of the University Medical Center Utrecht. All participants gave written informed consent at inclusion. Unrelated FCHL patients were recruited from the Lipid Clinic of the University Medical Center Utrecht. These subjects met the following criteria: a primary hyperlipidemia and at least one first degree relative with a different hyperlipidemic phenotype, at least twice elevated plasma apo B concentrations (>1.2 g l 1) before initiation of
Self-measurement of capillary TG (TGc) was performed with a TG-specific point-of-care testing device (Accutrend GCTR, Roche Diagnostics, Germany) [28 – 30]. Each participant always used the same device. Subjects were instructed to wash and dry their hands thoroughly before each measurement. With a lancing device, a drop of blood (30 Al) from the finger was obtained which was applied to the TG test strip in the TG analyzer. Subsequently, the TG concentration from the capillary blood sample was measured by a process of dry chemistry and colorimetry. If there was not enough blood on the TG test strip, subjects were advised to repeat the measurement. Each subject was
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instructed by the same investigator. The measurement range for capillary TG is 0.80 –6.86 mmol l 1. In a previous study, variation coefficients for different capillary TG concentrations were 3.3 – 5.3% [33]. The correlation coefficient between capillary TG measurements with the TG analyzer and plasma measurements according to enzymatic methods is 0.94 [22]. Similar results have been obtained in our laboratory [28,31,32]. Subjects were instructed to measure their capillary TG on three different days (preferentially Monday, Wednesday and Friday; not in the weekends) at the following time-points: fasting, before and 3 h after lunch and dinner and at bedtime. The 3-h postprandial measurements were performed exactly 3 h after the meals, regardless of intake of snacks. The results were recorded in a diary. Subjects were requested to refrain from heavy physical activity, although normal daily activities like bicycling to work were allowed. In addition, all participants were asked to continue their usual diet. In cases where there were one or more missing measurements during a day, the data for that particular day were not used for construction of an average diurnal TG profile. The mean diurnal TG profile was used for statistical analysis. 2.3. Analytical determinations Cholesterol and triglycerides were determined using a Vitros 250 analyzer (Johnson and Johnson Rochester, NY, USA). HDL was obtained from plasma after precipitation with dextransulphate/MgCl2. VLDL and LDL were isolated by ultracentrifugation as described [34]. Plasma apo B was measured by nephelometry using apo B monoclonal antibodies (Behring Diagnostics, OSAN 14/15). Plasma apo AI was measured by nephelometry using apo AI monoclonal antibodies (Behring Diagnostics, OUED 14/15). Glucose was measured by glucose oxidase dry chemistry (Vitros GLU slides) and colorimetry, and insulin was measured by competitive radio immunoassay with polyclonal antibodies. For estimation of insulin sensitivity, the HOMA index ( = glucose*insulin/22.5) was calculated.
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groups were tested with the Student’s t-test. In the case of TG, HOMA and insulin calculations were performed after logarithmic transformation because of the nonparametric distribution of these variables. Diurnal capillary TG profiles were calculated as mean integrated area under the TG curve (TGc-AUC) using Graphpad Prism version 3.0 (Graphpad Prism, San Diego, CA, USA). All calculations of TGc-AUC were preformed with the average of 2 or 3 days. Coefficients of variation (CV) were calculated using the formula: CV = S.D./mean*100 as described [28]. For statistical analysis, SPSS version 10.0 (SPSS, Chicago, IL, USA) was used. Statistical significance was reached when P < 0.05 (two-sided).
3. Results 3.1. Variability of fasting plasma lipids and apolipoproteins in FCHL and non-FCHL subjects (Tables 1 and 2) In Table 1, general characteristics and fasting biochemical parameters from 18 untreated FCHL subjects are compared to 16 healthy volunteers. There were no differences in gender distribution, age, BMI or glucose. Fasting plasma lipids, apolipoproteins and Table 1 General characteristics of 18 untreated FCHL subjects compared to 16 healthy controls matched for BMI, age and gender FCHL patients (n = 18)
Healthy controls (n = 16)
8/10 44.29 (10.65) 25.69 (2.59) 6.80 (2.09)** 3.06 (1.44)** 0.87 (0.25)** 3.62 (1.90)* 0.89 (0.69)** 1.33 (0.82)** 1.26 (0.22)** 1.20 (0.26)* 5.38 (0.49) 13.82 (5.94)** 3.28 (1.42)**
8/8 43.83 (7.97) 24.31 (2.68) 4.29 (0.87) 1.17 (0.35) 1.14 (0.28) 2.33 (0.80) 0.21 (0.12) 0.43 (0.22) 0.81 (0.20) 1.39 (0.21) 5.10 (0.34) 8.19 (3.80) 1.88 (0.96)
2.4. Statistics
Gender (f/m) Age (years) BMI (kg m 2) Plasma chol (mmol l 1) Plasma TG (mmol l 1) HDL-C (mmol l 1) LDL-C (mmol l 1) VLDL-C (mmol l 1) VLDL-TG (mmol l 1) Apo B (g l 1) Apo AI (g l 1) Glucose (mmol l 1) Insulin (mmol l 1) HOMA
Data are given as mean F S.D. in tables and as mean F S.E.M. in figures. Differences between
Data are given as mean F S.D. * P < 0.05. ** P < 0.01 compared to healthy controls.
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Table 2 Variability of fasting plasma concentrations calculated as coefficient of variation in FCHL and matched control subjects
Plasma chol Plasma TG HDL-C LDL-C VLDL-C VLDL-TG Apo B Apo AI
FCHL patients (n = 18) CV (%)
Healthy controls (n = 16) CV (%)
12.92 (11.40) 23.28 (10.22) 12.78 (8.18) 20.99 (21.91) 51.41 (25.40) 52.53 (41.46) 13.17 (15.78) 6.37 (5.20)
11.40 (9.14) 20.40 (8.22) 11.41 (4.31) 19.63 (18.45) 62.59 (28.53) 41.56 (17.27) 11.30 (10.61) 7.81 (4.56)
**P < 0.01 compared to healthy controls. The mean duration of follow-up was 48.91 F 35.46 days. Nine FCHL subjects were tested twice and nine were tested thrice. Controls were tested three times. Data are given as mean F S.D.
insulin were higher in FCHL subjects. The mean duration of follow-up was 48.91 F 35.46 days. Nine FCHL subjects were tested twice and nine were tested thrice during this period. Controls were tested three times. There were no significant differences in the coefficients of variation for any of the tested variables between FCHL subjects and controls (Table 2). The highest coefficient of variation was found for VLDLcholesterol en VLDL-triglycerides in FCHL and con-
Table 3 Variability of diurnal capillary TG measurements and total diurnal triglyceridemia (TGc-AUC) in FCHL and control subjects
Capillary TG Capillary TG Capillary TG Capillary TG Capillary TG Capillary TG TGc-AUC
fasting before lunch after lunch before dinner after dinner bedtime
FCHL patients (n = 17) CV (%)
Healthy controls (n = 90) CV (%)
36.26 25.14 23.73 21.16 32.66 26.02 15.89
24.87 26.45 25.86 25.45 27.71 30.54 15.13
(24.73) (24.73) (16.32) (14.21) (22.34) (18.61) (11.29)*
(17.24) (18.23) (16.77) (16.65) (17.61) (17.51) (10.98)*
* P < 0.01 compared to CV of fasting capillary TG.
trols and the lowest CV was found for plasma apo AI and apo B in both groups. 3.2. Variability of postprandial lipemia in FCHL and subjects (Fig. 1 and Table 3) In this part of the study, 90 healthy controls and 17 untreated FCHL subjects measured their capillary TG concentrations during 3 days in 1 week. Data from the 90 healthy controls have been published before [21]. Fig. 1 shows that at all time points, capillary TG was higher in FCHL than in controls, resulting in a higher total triglyceridemia estimated by the area
Fig. 1. Mean diurnal capillary TG concentrations at 6 different time points in 17 untreated FCHL subjects (n) compared to 90 healthy controls (.). Data are mean F S.E.M. Note: the error bars in the control group were so low that they are not visible at all the time points.
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under the TG curve (48.90 F 25.22 mmol h l 1 in FCHL and 20.11 F 6.79 mmol h l 1 in controls; P < 0.001). The coefficients of variation for each time point are given in Table 3. There were no significant differences between FCHL and controls at any time point. The CV for postprandial lipemia (TGc-AUC) was significantly lower in FCHL and controls compared to the CV of fasting capillary TG measurements.
4. Discussion This is the first study to systematically investigate the variability of fasting and postprandial lipemia during a relatively short time period in FCHL subjects not using lipid lowering drugs in comparison to normolipidemic healthy subjects. Until now, it has been reported by our group and many others that lipoprotein phenotypes were highly variable in FCHL patients. Our data suggest that whereas fasting plasma lipids may be highly variable, especially total TG and VLDL-associated lipids, the variability is not higher than in controls. In 1983, Brunzell et al. [22] were the first to describe that the lipoprotein phenotype in a single patient with FCHL can change from time to time. However, the authors did not investigate this systematically and it is not clear how they came to that conclusion. In a recent report [25] from the same group, 20-year prospective data on lipoprotein and apolipoprotein abnormalities in FCHL was described. One third of participants was discordant for hyperlipidemic status at baseline and 20-year follow-up (16.4% changed from normolipidemic to hyperlipidemic status and 17.1% changed from hyperlipidemic to normolipidemic status). Similar or higher degrees of discordance have been reported in individuals without known FCHL [26,27], which is in line with the present report demonstrating that the intra-individual variability of fasting plasma lipids of FCHL subjects and healthy controls is very similar. Our data suggest that changing phenotypes within one individual should not be used as a characteristic of the FCHL phenotype, at least within the time frame reported here. It should be noted that even the intra-individual variations of apolipoproteins and postprandial lipemia were similar to controls.
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In a recent report from our group [28], it was shown that diurnal triglyceridemia represented postprandial lipemia as estimated by standardized oral fat loading tests. In addition, it was suggested that the variability of the diurnal triglyceridemia as studied here was lower than the variability of fasting plasma TG. In the small groups of subjects described in the present report, this seems to be the case since the coefficient of variation of diurnal triglyceridemia was lower than that of fasting capillary triglycerides in both FCHL and controls (Table 3). In future trials aimed to modulate TG, it would be reasonable to use diurnal triglyceridemia as the target for treatment instead of fasting TG since diurnal triglyceridemia is a more stable parameter than fasting triglycerides. It is of interest that the highest CV was found for fasting VLDL-C and VLDL-TG and total fasting plasma TG. This may represent changes in hepatic VLDL production within the same individual. Several factors may be responsible for this variable hepatic VLDL output like hormones, diet and exercise. All these factors may influence the availability of hepatic triglycerides for VLDL synthesis, which has been reported to determine the phenotypic expression in FCHL subjects [35]. These factors may very well be acting in both FCHL and controls in a similar way since there was no difference in CV between both groups for fasting VLDL. Changes in insulin sensitivity may largely influence hepatic VLDL secretion and, therefore, plasma TG concentrations. It has also been shown that insulin sensitivity can change within subjects by 17% [36]. The variation of insulin sensitivity within FCHL subjects is not known, although it is well established that FCHL subjects are insulin resistant [14,16 –18,37– 40]. The patients described in this report were also insulin resistant as suggested by the significantly increased HOMA compared to controls. Although this study should be interpreted with caution due to the relatively small number of subjects and the relatively short time span of follow-up, the results are unexpected. A large prospective study is not feasible since it is not ethical to keep hyperlipidemic patients at risk without medication for a longer period of time. Changing lipoprotein phenotypes may be a general characteristic not exclusively associated with FCHL.
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Acknowledgements Roche Diagnostics (Mannheim, Germany) is greatly acknowledged for providing the Accutrend GCT meters with accessories.
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Intra-individual variations of fasting plasma lipids, apolipoproteins and postprandial lipemia in familial combined hyperlipidemia compared to controls D. Delawi, S. Meijssen, M. Castro Cabezas * Department of Vascular Medicine F02.126, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands Received 10 July 2002; received in revised form 30 October 2002; accepted 1 November 2002
Abstract Background: The intra-individual variability of plasma lipids and apolipoproteins has not been studied systematically in familial combined hyperlipidemia (FCHL). Methods: Intra-individual changes in fasting plasma lipids and apolipoproteins B and AI were determined in 18 untreated FCHL subjects and 16 unrelated, normolipidemic subjects. Participants were matched for gender, age and body mass index. The mean follow-up period of fasting plasma lipids was 48.91 F 35.46 (mean F S.D.) days. Postprandial lipemia was determined on 3 different days in 1 week in 90 healthy controls and 17 untreated FCHL subjects by the area under the diurnal capillary triglyceride curve (TGc-AUC). Results: The coefficients of variation (CVs) for fasting plasma TG were similar between FCHL (23.2 F 10.2%) and controls (20.4 F 8.2%). The CVs for HDL-C, apo B and apo AI were the lowest of all fasting plasma measurements in both groups and there was no significant difference between FCHL (12.8 F 8.2%, 13.2 F 15.8% and 6.4 F 5.2%, respectively) and controls (11.4 F 4.3%, 11.3 F 10.6% and 7.8 F 4.6%, respectively). The CVs for postprandial lipemia were not different between FCHL (15.9 F 11.3%) and controls (15.1 F 11.0%), and were significantly lower than the CV of fasting capillary TG (TGc) in the same period (36.3 F 24.7% and 24.9 F 17.2%, respectively). Conclusions: Our study does not provide evidence for short-term major changes in fasting or postprandial lipemia or apolipoproteins in FCHL when systematically compared to healthy controls. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Triglycerides; Familial combined hyperlipidemia; Atherosclerosis; Postprandial lipemia; Apolipoproteins
1. Introduction Familial combined hyperlipidemia (FCHL) is the most frequent dominantly inherited disorder of lipid metabolism leading to increased risk for atherosclerosis [1 –7]. The diagnosis is based on clinical criteria * Corresponding author. Tel.: +31-30-2507399; fax: +31-302518328. E-mail address: [email protected] (M. Castro Cabezas).
such as the presence of ‘‘multiple-type hyperlipidemia’’ [1,3,5,6] in relatives of index patients, increased concentrations of plasma apolipoprotein (apo) B and a positive family history of premature cardiovascular disease (CVD). The genetic basis of FCHL has not been elucidated, although several groups have provided evidence suggesting that different genes are involved in the pathogenesis of this disorder [8 – 10]. Several metabolic characteristics of FCHL have been described during the last 20 years. Patients with
0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0009-8981(02)00420-5
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FCHL are characterised by hepatic apo B overproduction [11 –13], delayed clearance of chylomicron remnants [14,15], insulin resistance [14,16,17], a primary defect in the metabolism of triglycerides and plasmafree fatty acids [14,18] with enhanced hepatic flux of free fatty acids resulting in exaggerated production of postprandial ketone bodies [19]. In addition, in a subset of patients, decreased activity of postheparin plasma lipoprotein lipase has been described [20,21]. One of the earliest described clinical characteristics was the presence of different lipoprotein phenotypes at one time point in members of a family unit [1,2,5]. This has been called ‘‘multiple-type hyperlipidemia’’. In addition, several investigators have observed that one individual patient may show different phenotypes during a longer period of time and have used the term ‘‘multiple-type hyperlipidemia’’ in this context. This was based on an early report by Brunzell et al. [22]. However, no systematic studies have been published on this subject and the metabolic basis for changing phenotypes in individual FCHL patients is not known. In non-FCHL subjects, fasting plasma lipids are widely variable [23,24]. The purpose of our study was to establish the variability of lipoprotein patterns in FCHL subjects on their regular diet. Since medication will affect the phenotypes, we evaluated the variation of the fasting lipoproteins of a group of FCHL index patients who were off medication for at least 1 month. In addition, the short-term variability of postprandial lipemia using diurnal capillary triglyceride profiles was evaluated and compared to healthy controls.
lipid lowering therapy and a positive family history of premature coronary heart disease (CHD) defined as myocardial infarction or cerebrovascular disease before the age of 60 in at least one blood-related subject of the index patient. In addition, the patients fulfilled the following inclusion criteria: absence of xanthomas, BMI < 30 kg/m2, absence of apo E2/E2 genotype, no use of drugs affecting lipid metabolism including lipid lowering drugs for at least 4 weeks before inclusion in this study and consumption of less than three units of alcohol/day. FCHL patients were on a low fat, low cholesterol diet. All FCHL patients participated in an ongoing study aimed to evaluate the effects of lipid lowering drugs on postprandial lipemia evaluated by oral fat loading tests. The results of the oral fat loading tests will be published separately. Normolipidemic, healthy controls (90) without a family history of cardiovascular disease or diabetes, absence of apo E2/E2 genotype and not using drugs known to affect lipid metabolism were recruited by advertisement. On the morning of inclusion, blood was collected after a 10-h fasting period for measurements of lipids, apolipoproteins, insulin and glucose. In addition, 16 different controls fulfilling the same inclusion criteria were selected to participate in the fat loading tests as matched healthy controls for the FCHL subjects. In order to be included, these controls needed to have the same anthropometric characteristics and age as the FCHL subjects. These 16 subjects visited our department on several occasions during a longer period of time and extensive fasting lipoprotein analysis was performed. 2.2. Capillary TG self-measurements
2. Subjects and methods 2.1. Subjects The study protocol was approved by the Human Investigations Review Committee of the University Medical Center Utrecht. All participants gave written informed consent at inclusion. Unrelated FCHL patients were recruited from the Lipid Clinic of the University Medical Center Utrecht. These subjects met the following criteria: a primary hyperlipidemia and at least one first degree relative with a different hyperlipidemic phenotype, at least twice elevated plasma apo B concentrations (>1.2 g l 1) before initiation of
Self-measurement of capillary TG (TGc) was performed with a TG-specific point-of-care testing device (Accutrend GCTR, Roche Diagnostics, Germany) [28 – 30]. Each participant always used the same device. Subjects were instructed to wash and dry their hands thoroughly before each measurement. With a lancing device, a drop of blood (30 Al) from the finger was obtained which was applied to the TG test strip in the TG analyzer. Subsequently, the TG concentration from the capillary blood sample was measured by a process of dry chemistry and colorimetry. If there was not enough blood on the TG test strip, subjects were advised to repeat the measurement. Each subject was
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instructed by the same investigator. The measurement range for capillary TG is 0.80 –6.86 mmol l 1. In a previous study, variation coefficients for different capillary TG concentrations were 3.3 – 5.3% [33]. The correlation coefficient between capillary TG measurements with the TG analyzer and plasma measurements according to enzymatic methods is 0.94 [22]. Similar results have been obtained in our laboratory [28,31,32]. Subjects were instructed to measure their capillary TG on three different days (preferentially Monday, Wednesday and Friday; not in the weekends) at the following time-points: fasting, before and 3 h after lunch and dinner and at bedtime. The 3-h postprandial measurements were performed exactly 3 h after the meals, regardless of intake of snacks. The results were recorded in a diary. Subjects were requested to refrain from heavy physical activity, although normal daily activities like bicycling to work were allowed. In addition, all participants were asked to continue their usual diet. In cases where there were one or more missing measurements during a day, the data for that particular day were not used for construction of an average diurnal TG profile. The mean diurnal TG profile was used for statistical analysis. 2.3. Analytical determinations Cholesterol and triglycerides were determined using a Vitros 250 analyzer (Johnson and Johnson Rochester, NY, USA). HDL was obtained from plasma after precipitation with dextransulphate/MgCl2. VLDL and LDL were isolated by ultracentrifugation as described [34]. Plasma apo B was measured by nephelometry using apo B monoclonal antibodies (Behring Diagnostics, OSAN 14/15). Plasma apo AI was measured by nephelometry using apo AI monoclonal antibodies (Behring Diagnostics, OUED 14/15). Glucose was measured by glucose oxidase dry chemistry (Vitros GLU slides) and colorimetry, and insulin was measured by competitive radio immunoassay with polyclonal antibodies. For estimation of insulin sensitivity, the HOMA index ( = glucose*insulin/22.5) was calculated.
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groups were tested with the Student’s t-test. In the case of TG, HOMA and insulin calculations were performed after logarithmic transformation because of the nonparametric distribution of these variables. Diurnal capillary TG profiles were calculated as mean integrated area under the TG curve (TGc-AUC) using Graphpad Prism version 3.0 (Graphpad Prism, San Diego, CA, USA). All calculations of TGc-AUC were preformed with the average of 2 or 3 days. Coefficients of variation (CV) were calculated using the formula: CV = S.D./mean*100 as described [28]. For statistical analysis, SPSS version 10.0 (SPSS, Chicago, IL, USA) was used. Statistical significance was reached when P < 0.05 (two-sided).
3. Results 3.1. Variability of fasting plasma lipids and apolipoproteins in FCHL and non-FCHL subjects (Tables 1 and 2) In Table 1, general characteristics and fasting biochemical parameters from 18 untreated FCHL subjects are compared to 16 healthy volunteers. There were no differences in gender distribution, age, BMI or glucose. Fasting plasma lipids, apolipoproteins and Table 1 General characteristics of 18 untreated FCHL subjects compared to 16 healthy controls matched for BMI, age and gender FCHL patients (n = 18)
Healthy controls (n = 16)
8/10 44.29 (10.65) 25.69 (2.59) 6.80 (2.09)** 3.06 (1.44)** 0.87 (0.25)** 3.62 (1.90)* 0.89 (0.69)** 1.33 (0.82)** 1.26 (0.22)** 1.20 (0.26)* 5.38 (0.49) 13.82 (5.94)** 3.28 (1.42)**
8/8 43.83 (7.97) 24.31 (2.68) 4.29 (0.87) 1.17 (0.35) 1.14 (0.28) 2.33 (0.80) 0.21 (0.12) 0.43 (0.22) 0.81 (0.20) 1.39 (0.21) 5.10 (0.34) 8.19 (3.80) 1.88 (0.96)
2.4. Statistics
Gender (f/m) Age (years) BMI (kg m 2) Plasma chol (mmol l 1) Plasma TG (mmol l 1) HDL-C (mmol l 1) LDL-C (mmol l 1) VLDL-C (mmol l 1) VLDL-TG (mmol l 1) Apo B (g l 1) Apo AI (g l 1) Glucose (mmol l 1) Insulin (mmol l 1) HOMA
Data are given as mean F S.D. in tables and as mean F S.E.M. in figures. Differences between
Data are given as mean F S.D. * P < 0.05. ** P < 0.01 compared to healthy controls.
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Table 2 Variability of fasting plasma concentrations calculated as coefficient of variation in FCHL and matched control subjects
Plasma chol Plasma TG HDL-C LDL-C VLDL-C VLDL-TG Apo B Apo AI
FCHL patients (n = 18) CV (%)
Healthy controls (n = 16) CV (%)
12.92 (11.40) 23.28 (10.22) 12.78 (8.18) 20.99 (21.91) 51.41 (25.40) 52.53 (41.46) 13.17 (15.78) 6.37 (5.20)
11.40 (9.14) 20.40 (8.22) 11.41 (4.31) 19.63 (18.45) 62.59 (28.53) 41.56 (17.27) 11.30 (10.61) 7.81 (4.56)
**P < 0.01 compared to healthy controls. The mean duration of follow-up was 48.91 F 35.46 days. Nine FCHL subjects were tested twice and nine were tested thrice. Controls were tested three times. Data are given as mean F S.D.
insulin were higher in FCHL subjects. The mean duration of follow-up was 48.91 F 35.46 days. Nine FCHL subjects were tested twice and nine were tested thrice during this period. Controls were tested three times. There were no significant differences in the coefficients of variation for any of the tested variables between FCHL subjects and controls (Table 2). The highest coefficient of variation was found for VLDLcholesterol en VLDL-triglycerides in FCHL and con-
Table 3 Variability of diurnal capillary TG measurements and total diurnal triglyceridemia (TGc-AUC) in FCHL and control subjects
Capillary TG Capillary TG Capillary TG Capillary TG Capillary TG Capillary TG TGc-AUC
fasting before lunch after lunch before dinner after dinner bedtime
FCHL patients (n = 17) CV (%)
Healthy controls (n = 90) CV (%)
36.26 25.14 23.73 21.16 32.66 26.02 15.89
24.87 26.45 25.86 25.45 27.71 30.54 15.13
(24.73) (24.73) (16.32) (14.21) (22.34) (18.61) (11.29)*
(17.24) (18.23) (16.77) (16.65) (17.61) (17.51) (10.98)*
* P < 0.01 compared to CV of fasting capillary TG.
trols and the lowest CV was found for plasma apo AI and apo B in both groups. 3.2. Variability of postprandial lipemia in FCHL and subjects (Fig. 1 and Table 3) In this part of the study, 90 healthy controls and 17 untreated FCHL subjects measured their capillary TG concentrations during 3 days in 1 week. Data from the 90 healthy controls have been published before [21]. Fig. 1 shows that at all time points, capillary TG was higher in FCHL than in controls, resulting in a higher total triglyceridemia estimated by the area
Fig. 1. Mean diurnal capillary TG concentrations at 6 different time points in 17 untreated FCHL subjects (n) compared to 90 healthy controls (.). Data are mean F S.E.M. Note: the error bars in the control group were so low that they are not visible at all the time points.
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under the TG curve (48.90 F 25.22 mmol h l 1 in FCHL and 20.11 F 6.79 mmol h l 1 in controls; P < 0.001). The coefficients of variation for each time point are given in Table 3. There were no significant differences between FCHL and controls at any time point. The CV for postprandial lipemia (TGc-AUC) was significantly lower in FCHL and controls compared to the CV of fasting capillary TG measurements.
4. Discussion This is the first study to systematically investigate the variability of fasting and postprandial lipemia during a relatively short time period in FCHL subjects not using lipid lowering drugs in comparison to normolipidemic healthy subjects. Until now, it has been reported by our group and many others that lipoprotein phenotypes were highly variable in FCHL patients. Our data suggest that whereas fasting plasma lipids may be highly variable, especially total TG and VLDL-associated lipids, the variability is not higher than in controls. In 1983, Brunzell et al. [22] were the first to describe that the lipoprotein phenotype in a single patient with FCHL can change from time to time. However, the authors did not investigate this systematically and it is not clear how they came to that conclusion. In a recent report [25] from the same group, 20-year prospective data on lipoprotein and apolipoprotein abnormalities in FCHL was described. One third of participants was discordant for hyperlipidemic status at baseline and 20-year follow-up (16.4% changed from normolipidemic to hyperlipidemic status and 17.1% changed from hyperlipidemic to normolipidemic status). Similar or higher degrees of discordance have been reported in individuals without known FCHL [26,27], which is in line with the present report demonstrating that the intra-individual variability of fasting plasma lipids of FCHL subjects and healthy controls is very similar. Our data suggest that changing phenotypes within one individual should not be used as a characteristic of the FCHL phenotype, at least within the time frame reported here. It should be noted that even the intra-individual variations of apolipoproteins and postprandial lipemia were similar to controls.
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In a recent report from our group [28], it was shown that diurnal triglyceridemia represented postprandial lipemia as estimated by standardized oral fat loading tests. In addition, it was suggested that the variability of the diurnal triglyceridemia as studied here was lower than the variability of fasting plasma TG. In the small groups of subjects described in the present report, this seems to be the case since the coefficient of variation of diurnal triglyceridemia was lower than that of fasting capillary triglycerides in both FCHL and controls (Table 3). In future trials aimed to modulate TG, it would be reasonable to use diurnal triglyceridemia as the target for treatment instead of fasting TG since diurnal triglyceridemia is a more stable parameter than fasting triglycerides. It is of interest that the highest CV was found for fasting VLDL-C and VLDL-TG and total fasting plasma TG. This may represent changes in hepatic VLDL production within the same individual. Several factors may be responsible for this variable hepatic VLDL output like hormones, diet and exercise. All these factors may influence the availability of hepatic triglycerides for VLDL synthesis, which has been reported to determine the phenotypic expression in FCHL subjects [35]. These factors may very well be acting in both FCHL and controls in a similar way since there was no difference in CV between both groups for fasting VLDL. Changes in insulin sensitivity may largely influence hepatic VLDL secretion and, therefore, plasma TG concentrations. It has also been shown that insulin sensitivity can change within subjects by 17% [36]. The variation of insulin sensitivity within FCHL subjects is not known, although it is well established that FCHL subjects are insulin resistant [14,16 –18,37– 40]. The patients described in this report were also insulin resistant as suggested by the significantly increased HOMA compared to controls. Although this study should be interpreted with caution due to the relatively small number of subjects and the relatively short time span of follow-up, the results are unexpected. A large prospective study is not feasible since it is not ethical to keep hyperlipidemic patients at risk without medication for a longer period of time. Changing lipoprotein phenotypes may be a general characteristic not exclusively associated with FCHL.
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Acknowledgements Roche Diagnostics (Mannheim, Germany) is greatly acknowledged for providing the Accutrend GCT meters with accessories.
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