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Original Studies |
Departments of Internal Medicine G02.228 (T.B.T., H.W.W., P.C.N.J.S., M.C.C., D.W.E., G.M.D.-T.) and Endocrinology (P.S.v.D., H.P.F.K.), University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
Address correspondence and requests for reprints to: Dr. G. M. Dallinga-Thie, Department of Vascular Medicine, G02.228, University Hospital Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: Gdalling{at}azu.nl
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Abstract |
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It is concluded that patients with adult-onset GH deficiency have increased levels of fasting and postprandial RLP-C and an impaired endothelial function as measured as FMD. Treatment with rh-GH resulted in a decrease of postprandial RLP-C concentration, thereby improving the postprandial atherogenic lipoprotein profile and improvement of endothelial function, however, the clearance of large chylomicron particles as reflected by RE remained disturbed.
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Introduction |
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Triglyceride (TG)-rich lipoproteins, particularly lipoprotein remnants, have been shown to be involved in atherogenesis (10, 11). Disturbances in postprandial lipoprotein remnant levels were found in patients with premature coronary atherosclerosis like familial combined hyperlipidemia, familial hypercholesterolemia, and type 2 diabetes (12, 13, 14). Al-Shoumer et al. (15) observed an increase in plasma TG levels in AGHD patients during daily regular meals, suggesting abnormalities in postprandial clearance of lipoprotein particles. No data are yet available on detailed analyses of postprandial remnant lipoproteins in AGHD patients.
In the present study, we used both the classical retinyl ester (RE) analysis and a new remnant lipoprotein method based on the immunoseparation principle to study remnant metabolism developed by Nakajima et al. (16). Apolipoprotein (apo) AI-containing and apo B100-containing particles were bound to a Sepharose gel coupled with specific monoclonal antibodies against Apo B100 and Apo AI. In the remaining supernatant fraction, remnant particles were found with only apo B48 or with apoB100/apoE. Lipoprotein remnants isolated with this method maintained their pathological properties in in vitro studies (foam cell formation, decreased endothelial dilatation) (17). Remnant lipoprotein particles, isolated in this way, were associated with endothelialdependent vasomotor function (17) and restenosis of coronary arteries after percutaneous transluminal coronary angioplasty (18). The clearance of remnant particles was also assessed by incorporation and analysis of exogenous vitamin A (REs) as core label for lipoprotein particles of intestinal origin. The suitability of vitamin A as a marker for chylomicrons and its remnants has been criticized (19, 20). Incorporation of RE occurs mostly in the late postprandial period, as reflected by the delayed postprandial RE response compared with apoB48 analysis in the very low-density lipoprotein (VLDL)/chylomicron fraction (21). Furthermore, RE label has been shown to exchange to other lipoprotein particles at later postprandial time points.
In this case-controlled intervention study, we investigated whether disturbances in postprandial lipoprotein metabolism were associated with the atherogenic lipoprotein profile observed in AGHD patients and whether rh-GH treatment was capable of improving the atherogenic profile, thereby decreasing the risk for coronary artery disease in AGHD patients.
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Methods |
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Patients with adult-onset GH deficiency were recruited from the outpatient clinic of the Department of Endocrinology, University Hospital Utrecht. All patients had acquired GH deficiency in adult life due to recent (within 1 yr) treatment of a pituitary adenoma with surgery and/or radiotherapy. Other deficient pituitary hormones were supplemented for at least 6 months and were at a stable level at the start of the study. GH deficiency was defined as a peak plasma GH concentration less than 5 µg/L after the arginine infusion test. rh-GH was substituted to plasma insulin-like growth factor I (IGF-I) levels within the age-related normal range (22, 23). Exclusion criteria were presence of lipoprotein disorders such as familial hypercholesterolemia and familial combined hyperlipidemia, body mass index (BMI) more than 30, renal and/or liver disease, diabetes mellitus, Apo E2/E2 genotype, and a positive family history of premature atherosclerosis. Eight healthy control subjects, matched for age, gender, BMI, and ApoE genotype were selected for this study by advertisement. They had no diabetes; no hepatic, renal, thyroid, or cardiac dysfunction; and a negative family history for cardiovascular disease. Postheparin lipoprotein lipase (LPL) and hepatic lipase (HL) activities were measured at baseline in controls and before and after 6-month rh-GH treatment in AGHD patients. Oral fat load tests with RE were performed on a separate day.
The human investigation review committee of the University Hospital Utrecht approved this protocol, and written informed consent was obtained from all participants.
Oral fat loading vitamin A test
After an overnight fast (12 h), participants were admitted to the metabolic ward at 0730 h. Cream [consisting of 40% fat (wt/vol)] with a polyunsaturated fat to saturated fat ratio of 0.06, 0.001% cholesterol (wt/vol), and 2.8% carbohydrates (wt/vol)) were given as a single fat load in a dose of 50 g fat per m2 body surface area. After ingestion of the cream supplemented with 60.000 IU aqueous vitamin A per 125 mL cream, 10 hourly venous blood samples were collected from an indwelling catheter in the antecubital vene into EDTA-containing tubes. All blood samples, protected from light, were immediately put on ice, centrifuged, and analyzed. During the postprandial period, the subjects were allowed to drink only water or tea without sugar. None of the subjects experienced gastrointestinal complaints after drinking the cream.
Laboratory measurements
Plasma was obtained by centrifugation at 3000 rpm for 15 min at 4 C. TG and cholesterol were measured with a colorimetric assay (Monotest cholesterol kit no. 237574 and GPO-PAP no. 701912; Roche Diagnostics, Basel, Switzerland). Cholesterol was analyzed in the high-density lipoprotein (HDL) fraction isolated by the heparin-MnCl2 dextran-sulfate precipitation method (24). LDL cholesterol was calculated with the Friedewald formula (25). Plasma apo B concentrations were analyzed automatically on a Cobas Mira autoanalyzer (Unimate 3 Apo B; Roche Diagnostics). Plasma Apo E and Apo CIII concentrations were determined with a commercial test kit using the immunoelectrophoresis technique (Sebia Inc., Issy-les Moulineaux, France). The coefficient of variance (CV) for plasma Apo E and Apo C III analysis was less than 7.5%. The plasma insulin and IGF-I concentrations were determined with a RIA (26). Apo E genotype was determined as described by Dallinga-Thie et al. (27). Plasma for LPL and HL was obtained 20 min after an iv injection of 50 IU/kg heparin. Postheparin LPL and HL activity were assayed as described previously (28, 29). Nonesterified fatty acids [expressed as nmol free fatty acids min-1 (mU)/mL] were measured with an enzymatic assay (WAKO Chemicals, Neuss, Germany). HOMA-index (fasting glucose1fasting insulin/22.5) was calculated to estimate the insulin sensitivity. Body composition was assessed with bio-impedance analysis.
Assessment of lipoprotein remnants
Lipoproteins were separated by flotation using a single ultracentrifugation step in a Sf > 1000 fraction that contains chylomicrons, large chylomicron remnants, and large hepatic TG-rich lipoproteins, and a remaining infranatant fraction (Sf < 1000) containing small chylomicron remnants and all the other lipoproteins (30, 31). RE concentrations in plasma and in the Sf > 1000 and Sf < 1000 fraction were measured with high-performance liquid chromatography, as described by Ruotolo et al. (32). Recoveries of REs in the Sf > 1000 and Sf < 1000 were between 80% and 105%.
The remnant-like particle (RLP) fraction was prepared using an immunoseparation technique described by Nakajima and colleagues (16, 33). Briefly, 5 µL serum were added to 300 µL mixed immunoaffinity gel suspension containing monoclonal antihuman apo A-I (H-12) and antihuman apo B-100 (JI-H) antibodies (Japan Immunoresearch Laboratories, Takasaki, Japan). The reaction mixture was gently shaken for 120 min at room temperature, followed by standing for 15 min. Then, 200 µL of the supernatant was withdrawn for the assay of RLP-C and RLP-TG. Cholesterol (CV, <3%) and TGs (CV, <3%) in the RLP fraction were measured by an enzymatic assay using an automatic chemistry analyzer (ABX Diagnostics, Montpellier, France).
Forearm vasomotion study
Before the test meal blood samples were obtained for baseline values, and a forearm vasomotion test was performed. The ultrasound measurements were performed in a supine position at the elbow of the right arm using a vessel wall-movement system (Wall Track System; Pie Medical, Maastricht, The Netherlands), consisting of an ultrasound imager with a 7.5-MHz linear array transducer connected to a data acquisition system and a personal computer. In short, an optimal twodimensional B-mode image of the brachial artery was obtained. An M-line perpendicular to the vessel was selected. Next, the ultrasound system was switched to M-mode, after which storage of data started. The vessel movement detector system repeatedly registered end-diastolic vessel diameter during a period of five to six cardiac cycles. This procedure was performed three times. The measurements were averaged to provide for a baseline diameter measurement. By inflation of a blood pressure cuff for 4 min at a pressure of 100 mm Hg above the systolic blood pressure, ischemia was applied to the forearm distal to the location of the transducer. Ultrasonography continued for 3 min after cuff release with measurements at 30-sec intervals. The widest lumen diameter was taken as a measure for maximal diameter. Measurements were obtained for another 5 min, at 1-min intervals. Flow-mediated dilatation was expressed as a percentage change relative to baseline diameter. The intersession variability was 3.4%.
Statistical analysis
Data are presented as means ± SD, unless shown otherwise. Area under the integrated curve (AUC) was calculated using data from the first 8 h after the start of the oral fat loading test for postprandial TG, RE, and RLP-C using GraphPad Prism software (version 3.1; GraphPad Software, Inc., San Diego, CA). Effects of rh-GH substitution in AGHD patients and differences between untreated AGHD patients and controls were analyzed by two-tailed unpaired Student’s t tests. Pearson’s correlation or Spearman’s rank correlations were applied to evaluate relationships between parameters. A two-sided P value of 0.05 was considered significant. Statistical analysis was performed with GraphPad InStat (version 3.00 for Windows 95; GraphPad Software, Inc.).
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Results |
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Table 1
shows characteristics of the
subjects. The average duration of hypopituitarism was 40 ± 8
months. All eight patients were substituted with
T4, six patients with hydrocortisone, two
patients with cortisone acetate, seven patients with sex hormones
(males, testosterone esters; females, cyclic estrogen and
progesterone), and seven patients with desmopressin. Seven patients had
an apo E3/E3 genotype, and one patient had an apo E3/E4 genotype. All
patients were treated with rh-GH during minimally 6 months with an
average of 7 ± 1 month.
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Postprandial TG responses
After the oral fat load, maximum postprandial plasma TG levels
were reached at 3 h in control subjects and between 4 and 5 h
in AGHD patients (Fig. 1).
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). After correction for baseline
TG levels (incremental AUC-TG), this difference disappeared (8.62
± 5.92 mmol1h/L vs. 4.71 ± 2.42 mmol1h/L). Treatment
with rh-GH did not result in improvement fasting plasma TG levels.
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Fasting RLP-C concentrations were significantly elevated in AGHD
patients (0.29 ± 0.14 mmol/L) compared with in control subjects
(0.19 ± 0.06 mmol/L; P < 0.05; Fig. 2A). The maximum postprandial RLP-C
concentration was reached between 2 and 4 h in control subjects
and between 4 and 6 h in AGHD patients and was significantly
higher in AGHD patients. The AUC-RLP-C (Table 2
) and the incremental
AUC-RLP-C were significantly elevated in AGHD patients (4.46 ±
2.0 mmol1h/L, respectively, 2.13 ± 1.60 mmol1h/L) than in control
subjects (2.59 ± 1.08 mmol1h/L, respectively, 1.05 ± 0.72
mmol1h/L; P < 0.05). GH treatment resulted in a
significant decrease of AUC-RLP-C and incremental AUC-RLP-C (Table 2),
suggesting an improvement of postprandial clearance of RLP after
normalization of the GH axis, although the peak time for RLP-C was
unchanged (Fig. 2). It is important to note that despite the fact that
a strong correlation existed between baseline TG and RLP-C, they seemed
to have different metabolic properties in AGHD patients. In AGHD
patients, positive correlations were observed for incremental AUC-RLP-C
and baseline plasma cholesterol concentrations (r = 0.63;
P < 0.05), plasma TG concentrations (r = 0.62;
P < 0.05), LDL cholesterol (r = 0.62;
P < 0.05), and Apo B concentrations (r =
0.63;P < 0.05), whereas IGF-I concentrations showed a
negative correlation (r = -0.54, P < 0.03).
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Maximum postprandial plasma RE concentrations were reached at
4 h in control subjects and between 5 and 8 h in AGHD
patients and were higher, albeit not statistically significant, in AGHD
patients (Fig. 3A). Similarly the area
under the RE curve (AUC-RE) was higher in AGHD patients (Table 2) but
the difference did not reached the level of statistical significance.
GH treatment resulted in even higher maximal postprandial RE levels,
and the difference with the control subjects now reached the level of
significance (P < 0.05). The AUC-RE in the Sf <
1000 fraction increased after rh-GH treatment in AGHD patients and was
significantly elevated compared with control subjects
(P < 0.05; Table 2).
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To assess the atherosclerotic burden of the AGHD patients, we
measured the flow-mediated diameter of the brachial artery. No
measurements were performed in control subjects. Rh-GH treatment in
AGHD patients resulted in an increase in the flow-mediated diameter in
the brachial artery as shown in Fig. 4
(from 5.9 ± 3.3% to 10.2 ± 4.0%, P <
0.05). The basal diameter of the artery was similar in AGHD patients
before treatment: 4.4 ± 0.8 mm and after rh-GH treatment:
4.3 ± 0.7 mm.
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Discussion |
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Postprandial remnant lipoprotein particles have to be considered important mediators into the atherogenic process (34, 35). We used two different approaches to assess lipoprotein remnant metabolism. A new isolation method based on immunoseparation of remnant lipoproteins with Sepharose coated with specific antibodies against apo B100 and Apo AI was applied, resulting in the scavenging of all HDL and most of the LDL and VLDL particles from the plasma. In the supernatant fraction the RLP (apo B48-containing and apo B100/Apo E-enriched particles) were recovered. Analysis of postprandial RLP consisting mostly of Apo B48 particles is in agreement with assessment of postprandial apo B48 in VLDL/intermediate density fractions (Dallinga-Thie, G. M., unpublished data). Furthermore, the classical analysis of vitamin A (RE), which is incorporated into the core of the newly synthesized chylomicron particle, was used. Assessment of RE concentrations in the plasma and in the Sf < 1000 and Sf > 1000 fractions over a period of 8 h will provide evidence for in vivo chylomicron remnant clearance.
Our results show for the first time that in AGHD patients abnormalities in postprandial response were characterized by decreased clearance of RLP-C and RE in the Sf < 1000 fraction, whereas fasting levels of RLP-C were also significantly increased. Treatment with rh-GH resulted in significant improvement of RLP-C clearance, but no effects were observed in RE metabolic behavior and in fasting plasma RLP-C. Incorporation of vitamin A occurred mostly at later time points during the postprandial phase into larger chylomicron particles (36). After ingestion of dietary fatty acids the volume of the newly formed chylomicron particles increased, thereby improving the capacity to carry vitamin A. Larger chylomicrons and its remnants were considered to be less atherogenic particles than smaller, early postprandial remnants (35). It has been shown that in rat enterocytes the secretion of chylomicrons occurred in a bimodal way. De novo-secreted chylomicrons in the late postprandial period and the continuous secretion of smaller chylomicrons predominantly in the fasting and early postprandial period, albeit some degree of secretion of smaller particles remained throughout the later period. We hypothesize that apo B 48 and RLP-C reflects particles with identical behavior, whereas RE marks the properties of intestinal postprandial lipoprotein particles with a different metabolic behavior.
Our results suggest that the secretion of large chylomicron particles remained abnormal after GH substitution. GH therapy resulted in an up-regulation of the hepatic expression of the LDL receptor (37), resulting in improved clearance of apo B-containing lipoprotein particles. In fact, we observed a significant decrease in plasma LDL-cholesterol level and concomitant decrease in VLDL-cholesterol. Both apo B100- and apo B48-containing particles share the same degradation pathways (38), involving either the LDL receptor pathway or alternative pathways involving the LDL receptor-related protein and proteoglycans. The preferential improvement of only the RLP-C fraction is in support of a role of the LDL receptor pathway in apo B48 remnant particle clearance. No correlations were found between changes in LPL activity and postprandial parameters. It has been reported that GH supplementation results in a specific decrease of LPL activity in adipose tissue but not in a change in LPL activity in skeletal muscle tissue (39, 40), which has been shown to be correlated with a beneficial lipoprotein profile.
We hypothesize that fasting plasma RLP-C levels resembles the presence of circulating apoB100 remnant particles enriched with apoE. Due to conformational changes in the particles they do not bind to the monoclonal antibody and remained in the supernatant fraction (16). The fact that its concentration remained elevated after GH treatment is in favor for a different metabolic behavior of these remnant particles compared with apo B48-containing remnants. Additional studies are required to dissect the metabolic behavior of these different remnant fractions.
Increased cardiovascular mortality due to accelerated
atherosclerosis is a clinical feature in adult-onset GH deficiency
(1, 2, 3). Impaired endothelial-dependent vasodilatation in
response to flow is associated with early atherogenesis (41, 42). Different interventions in the atherosclerotic process,
including GH substitution in adult-onset GH deficiency, have been shown
to directly modulate endothelial functions (43, 44, 45). In
the present study, a significant increase in flow-mediated dilatation
after treatment with rh-GH was observed, which is in agreement with an
improvement of the atherogenic profile in response to GH treatment in
AGHD patients (4). It has been recognized that RLP-C
offered independent assessment for coronary heart disease risk
in addition to TG. Incubation with an isolated RLP fraction reduced
endothelial-dependent vasorelaxation in vitro, which could
be reversed by intervention with 
-tocopherol (46, 47).
In humans, plasma RLP-C concentrations were negatively associated with
plaque regression of the coronary artery (17).
In conclusion, we showed a significant decrease in postprandial RLP-C concentration and improvement of the endothelial function in AGHD patients after GH replacement therapy. We were not able to show a direct relationship between the improved endothelial function and improvement of the plasma lipoprotein profile, probably due to the small sample size. Improved postprandial lipoprotein remnant levels and improved endothelial function reflect a less atherogenic state and support the beneficial effect of early GH replacement therapy in AGHD patients.
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Acknowledgments |
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Footnotes |
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Received May 17, 2000.
Revised August 21, 2000.
Accepted September 7, 2000.
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References |
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), AGHD patients with rh-GH treatment
(
), and control subjects (
). Data are presented as mean ±
SEM.
