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Department of Metabolic Medicine, St. Mary’s Hospital, Imperial College of Science, Technology, and Medicine, Paddington, London, United Kingdom
Address all correspondence and requests for reprints to: Tara Kearney, M.D., Department of Metabolic Medicine, St. Mary’s Hospital, Imperial College of Science, Technology, and Medicine, Paddington, London, United Kingdom. E-mail: t.kearney{at}ic.ac.uk
Abstract
The dyslipidemia associated with hypopituitarism may contribute to increased vascular mortality. The atherogenic potential of lipoproteins is determined not only by concentration but also by their composition. We therefore studied very low-density lipoprotein composition and apolipoprotein B kinetics in 16 hypopituitary subjects and 16 controls. Hypopituitarism was associated with reduced high-density lipoprotein cholesterol (0.98[0.82–1.18] vs. 1.35[1.15–1.41] mmol/liter, P < 0.001) and increased triglyceride concentrations (1.64[1.09–2.77] vs. 1.12[0.66–1.67] mmol/liter, P = 0.01). Total (P = 0.76) and low-density lipoprotein cholesterol (P = 0.56) concentrations were similar. Very low-density lipoprotein- triglyceride was significantly increased (1.48[1.02–2.55] vs. 0.9[0.31–2.30] mmol/liter, P = 0.004), but very low-density lipoprotein cholesterol levels were similar (P = 0.93). The molar ratios of very low-density lipoprotein-triglyceride:apolipoprotein B (6193[4283–9566] vs. 3599[3188–6854], P = 0.005) and very low-density lipoprotein-triglyceride:cholesterol (2.8[1.98–3.78] vs. 1.6[1.44–2.80], P < 0.003) were significantly increased; very low-density lipoprotein-cholesterol:apolipoprotein B molar ratios (P = 0.93) were similar. Very low-density lipoprotein apolipoprotein B fractional synthetic rate (a measure of apolipoprotein B catabolism, P = 0.42) and pool size (P = 0.63) were similar. The very low-density lipoprotein apolipoprotein B absolute synthetic rate (a measure of apolipoprotein B synthesis) tended to be higher in hypopituitarism (17.7[2.91–19.50] vs. 26.6[19.64–28.05] mg/kg per day, P = 0.24) but failed to reach statistical significance. The absolute synthetic rate, and hence very low-density lipoprotein production, correlated with very low-density lipoprotein triglyceride:apolipoprotein B ratio (P = 0.02, Rs = 0.63), suggesting that triglyceride enrichment of very low-density lipoprotein is important in the mechanism underlying very low-density lipoprotein overproduction in hypopituitarism. Because triglyceride-enriched lipoproteins are proatherogenic, this may contribute to the vascular mortality observed in hypopituitarism. The reasons for these observations are unknown; GH deficiency or routine endocrine replacement may be important.
SEVERAL STUDIES HAVE demonstrated increased cardiovascular mortality in hypopituitary subjects receiving conventional endocrine replacement. The reasons for this are unknown, although the dyslipidemia observed with GH deficiency may be important. Most (1, 2, 3, 4) but not all (5) studies have demonstrated higher total and low-density lipoprotein (LDL) cholesterol concentrations. High-density lipoprotein (HDL) cholesterol levels have been normal (6) or low (1, 2, 7). Very low-density lipoproteins (VLDL) levels have been increased (1, 2, 3, 4, 5, 8). Apolipoprotein B (apoB) 100, the major lipoprotein of VLDL and LDL, has been increased in some (1, 2, 3, 4, 6, 9, 10, 11) but not all (7) studies. Increasing emphasis is placed on the composition of lipoproteins. It is now well recognized that triglyceride-enriched LDL (small, dense LDL) is strongly atherogenic (12, 13). Evidence is also mounting to suggest that triglyceride-laden VLDL may be atherogenic (14, 15). Conversely, the triglyceride enrichment of VLDL seen in lipoprotein lipase deficiency is not associated with an increase in atherosclerosis (16). Christ et al. (17) previously examined the lipid content of VLDL and reported an increase in VLDL cholesterol content in hypopituitary subjects. However, this has not been substantiated in other studies (18).
Few studies have examined the effects of hypopituitarism on VLDL apoB kinetics (9, 11, 18). All have used stable isotope techniques to determine the production and catabolic rates of VLDL apoB. Hypopituitarism has been associated with increased VLDL apoB production in all studies. The fractional synthetic rate (equal to the fractional catabolic rate in steady-state conditions) in hypopituitary subjects was reduced in two studies (9, 11) but not in the third (18).
The reasons for these discrepancies are unclear, although the previous studies were of relatively small numbers of patients and controls. In the first study, six of the seven hypopituitary subjects were dyslipidemic (total cholesterol of greater than 5.5 mmol/liter or plasma triglyceride of greater than 2.3 mmol/liter, mean total cholesterol of 6.6 ± 0.3 mmol/liter). Although subjects were randomly selected in both studies, in the study performed by our own group (Chrisoulidou et al.) (18), hypopituitary subjects were normolipidemic (mean total cholesterol of 5.21 ± 0.3 mmol/liter). These differences in lipid profile may be responsible for some of the discrepant results; it is possible that lipid concentration and kinetic abnormalities are observed only in those with a more profound dyslipidemia. Studies of this nature are limited; to clarify this issue, we have therefore studied a larger group of randomly selected subjects with hypopituitarism. Previous authors have suggested that the metabolism of VLDL may be determined not only by the plasma concentrations but also by the composition of VLDL; Packard et al. (19) demonstrated that triglyceride-enriched VLDL may be preferentially metabolism by the hepatic receptor, rather than by delipidation to LDL. We therefore aimed to study the relationship between VLDL composition and apoB kinetics.
Subjects and Methods
Subjects
Sixteen GH-deficient hypopituitary subjects were recruited from
the endocrine clinic at St. Mary’s Hospital (London, UK). The study
was open to hypopituitary subjects who had no active pituitary disease
or other medical complications. Subjects received optimal conventional
hormone replacement in accordance with normal clinical practice and had
been stable on this treatment for at least 1 year. In particular,
thyroxine replacement was optimized to ensure normal T3 concentrations.
Initial lipid or lipoprotein concentrations were not included as
criterion for inclusion in the study. Two postmenopausal women did not
receive sex hormone replacement. All patients had a stimulated GH
response to hypoglycemia (performed in 15 subjects) or glucagon
(performed in 1 subject) of less than 6 µl, confirming GH
deficiency. Subjects did not have any other medical conditions. The
causes and treatment of hypopituitarism are shown in Table 1
.
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. The study received
ethical approval from the Parkside Health Authority, and all subjects
gave written informed consent.
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Isotopes. 1-13C-Leucine (99%) was obtained from Cambridge Isotope Laboratories (Woburn, MA). It was dissolved in 150 mmol/liter sodium chloride, packaged in 5-ml glass ampoules (10 ml/liter) by the department of chemical pathology at the Royal Free Hospital, London, and tested for pyrogenicity and sterility.
Study protocol. Patients and controls attended the Metabolic Day Ward at St. Mary’s Hospital at 0745 h following a 10-h overnight fast. During the study, subjects were allowed water only. A meal was given on completion of the infusion. Iv cannulae were positioned in each antecubital fossa, one for the stable isotope infusion, the other for sampling. Fasting samples were taken for estimation of total cholesterol, triglyceride, HDL cholesterol, and glucose. Blood for estimation of testosterone (where appropriate), routine hematology, renal, thyroid, and liver function tests were taken in accordance with normal clinical practice.
A primed (2 mg/kg), constant (2 mg/kg per h) infusion of
1-131C-leucine was administered for 8 hours,
using a Baxter automatic pump. Samples were taken before the initiating
the infusion and hourly thereafter. At each time point, 5 ml of blood
were drawn into heparinized tubes. The plasma was immediately separated
by low-speed centrifugation (2000 rpm) at 4 C for 30 min and stored at
-70 C for analysis of the 131C enrichment of the
-ketoisocaproic acid (-KIC), the deamination product of leucine.
At the same time points, 12 ml of blood was drawn into tubes in which
120 µl of 10% EDTA was added for the separation of lipid fractions
and subsequent isolation and analysis of 131C
enrichment of VLDL apoB100. Plasma from these samples was separated
immediately, as above, and transferred to ultracentrifugation tubes
(Beckman Coulter, Inc.), and 25 µl each of 5%
sodium azide (antifungal) and 5% gentamicin (bactericidal) was then
added. Plasma (0.5 ml) was stored immediately at -70 C for later
estimation of nonesterified fatty acids levels (NEFA). The volume of
plasma remaining at each time point was noted. The centrifuge tubes
were then filled completely with 150 mmol/liter saline in preparation
of ultracentrifugation performed on completion of the infusion.
VLDL separation.VLDL was separated by ultracentrifugation at densities of 1,006 g/ml for 18 h at 160,000 x g, with a Bromma 2,330-ultraspin centrifuge and an SRP 50AT rotor (LKB, Bromma, Gothenburg, Sweden) according to the method of Havel et al. (20) and delipidated using a mixture of ether/methanol (3:1 vol/vol). VLDL protein (50 µl) was subjected to PAGE (5–15%), the apoB band was excised and hydrolyzed in 2.0 ml of 6 N HCl at 110 C for 24 h, with 9 µg of Norleucine as the internal standard. The hydrolysate was dried under nitrogen, reconstituted in 0.5 ml 50% acetic acid and transferred to freshly prepared AG 50W-X8 cationic resin columns (Bio-Rad Laboratories, Inc., Hercules, CA). After washing with deionized water, the amino acids were eluted with 3-mol/liter NH4OH into glass reactivials (Wheaton, Mays Landing, NJ) and dried under nitrogen. The amino acid residues were reacted with acetonitrile and N-methyl-N-(tert-butidylmethylsilyl)-trifluoracetamide to form the bis (tert-butyldimethylsilyl) derivative.
Derivatization of -KIC
Isotopic enrichment of -KIC was determined using the method
of Ford et al. (21). -Ketovaleric acid
internal standard solution (50 µl) was added to 100 µl of plasma;
this was deproteinized with 1 ml of ethanol. After centrifugation, the
supernatant was decanted into reactivials and evaporated to dryness
under nitrogen. The residue was dissolved in phenylenediamine solution
(0.2% wt/vol) and deionized water. The coupled ketoacids were
extracted with ethyl acetate and dried over sodium sulfate. The dried
residue was derivatized with 50 µl of acetonitrile and 50 µl
N,O-bis (trimethylsilyl) trifluoroacetamide.
Measurement of stable isotope enrichment
Enrichment of both leucine and -KIC was quantified using a
Varian 3400 gas chromatograph/Finnigan Incos XL mass spectrometer
(Thermoquest, Hemel Hempstead, UK) in electron impact mode under
computer control. Selective ion monitoring of the derivatized samples
at m/z 302 for unlabeled leucine, m/z 303 for labeled leucine, m/z 232
for unlabeled -KIC, and m/z 233 for labeled -KIC was used to
determine isotopic abundance. The atom per excess (APE) enrichment of
both leucine and -KIC was calculated with a method equivalent to
that of Cobelli et al. (22) using the
formula:
Enrichment (APE) = (IRt –IR0/IRt –IR0 +100]) x 100
IRt = isotopic enrichment at time ‘t’
IRo = isotopic enrichment before start of stable isotope infusion.
The raw APEs of plasma leucine and -KIC enrichments were converted
to mole per excess by the application of a calibration curve obtained
by regression analysis of the plot of the theoretical MPE against
observed APE.
Calculation of the apoB production rate
The fractional synthetic rate (FSR) of VLDL apoB (pool/h) was determined by fitting the monoexponential function to the enrichment data:
E (t) = P (1-e-k (t-d)) where E (t) = the enrichment at time t,
P = plateau enrichment (-KIC precursor
enrichment)
K = FSR of VLDL apoB
D = intrahepatic delay time
The absolute synthetic rate (ASR) of VLDL apoB was calculated as the product of FSR and pool size. The latter was calculated by multiplying the plasma volume and the VLDL apoB concentration (23). The plasma volume was assumed to be 4.5% of the total body weight (kg). Some studies have suggested that GHD subjects may have reduced plasma volume. However, because this deficit is in the order of 0.5 l, this is unlikely to introduce any significant error in this calculation. VLDL apoB concentration was measured using a commercial kit employing an immunoturbidimetric method (Alpha Laboratories, Eastleigh, UK) on a centrifugal analyzer (COBAS-BIO, Roche Diagnostics, East Sussex, UK). All samples were assayed in triplicate. The inter- and intra-assay coefficients of variation were 5% and 7%, respectively.
Other assays
Total cholesterol, triglyceride, and HDL cholesterol were measured enzymatically using an AU 5200 analyser (Olympus Corp., Eastleigh, UK). Fasting VLDL cholesterol (24), VLDL triglyceride, and NEFA levels (25) were measured enzymatically using a centrifugal analyzer (COBAS-BIO, Roche Diagnostics). VLDL triglyceride and cholesterol content could not be determined in four subjects owing to insufficient sample.
Statistical analysis
Normally distributed data (age, sex, body mass index [BMI]) are described using the mean ± SEM. All other data are described using the median ± interquartile range. Parametric data are compared using an unpaired t test; nonparametric data are compared using the Mann-Whitney U test. Statistical significance was assumed at a 5% level. Spearman’s rank (Rs) correlation test was to determine the strength of relationship between nonparametric data. VLDL triglyceride to apoB molar ratios were calculated assuming a mean triglyceride molecular weight of 844.96 and an apoB molecular weight of 550,000.
Results
Control subjects and patients were matched for age (43 ± 3
vs. 43 ± 3 yr, P = 0.97), sex (nine
males and seven females in both groups) and BMI (28 ± 1
vs. 28 ± 1 kg/m2, respectively,
P = 0.77). Circulating lipid levels and kinetic data in
control and hypopituitary subjects are shown in Tables 3 and 4,
respectively.
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Patients and controls had similar total cholesterol levels (patients 5.23[4.20–6.23] vs. controls 5.53[4.42–6.64] mmol/liter, respectively, P = 0.79) and LDL cholesterol levels (patients 3.16[2.33–4.41] vs. controls 3.47[2.90–4.46] mmol/liter, P = 0.56). The hypopituitary group had significantly lower HDL cholesterol levels (0.98[(0.82–1.18] vs. 1.35[1.15–1.41] mmol/liter, P = 0.001) and significantly higher triglyceride levels (1.64[1.09–2.77] vs. 1.12[0.66–1.67] mmol/liter, P = 0.01).
Glucose tolerance and NEFA concentrations (Table 5)
NEFA levels were similar in the two groups (0.50[0.40–0.52] vs. 0.58[0.48–0.98] mmol/liter, hypopituitary vs. controls, respectively, P = 0.24). Fasting glucose concentrations were normal in controls (4.45[3.95–4.8]mmol/liter) and hypopituitary subjects (4.75[4.3–5.10]mmol/liter), with no statistical difference between the two groups (P = 0.64).
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VLDL lipid content in control and hypopituitary subjects is
shown in Tables 6 and 7, respectively. VLDL triglyceride was
significantly higher in hypopituitary subjects (1.48[1.02–2.55]
vs. 0.9[0.31–2.30] mmol/liter of VLDL, P
= 0.004), but VLDL- cholesterol levels were similar
(0.51[0.40–0.97] vs. 0.55[0.23–1.11] mmol/liter of
VLDL, P = 0.93). The VLDL triglyceride to apoB molar
ratio (6193[4283–9566] vs. 3599[3188–6854],
P = 0.005) and the VLDL triglyceride to
VLDL-cholesterol ratio (2.82[1.98–2.78] vs.
1.62[1.44–2.80], P = 0.0025) were significantly
higher in patients than controls. The VLDL cholesterol to apoB
molar ratios (2193[1451–3594] patients vs.
2395[1339–4347] controls, P = 0.89) were
similar.
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There were no significant differences in VLDL apoB FSR (controls 0.37[0.19–0.49] vs. patients 0.34[0.22–0.52] pools/h, P = 0.42) or in pool size (controls 1.93[1.48–4.06] vs. patients 2.18[1.72–3.53] mg/kg , P = 0.63). Although it tended to be higher in the hypopituitary patients, the VLDL apoB ASR (controls 17.7[2.91–19.50] vs. patients 26.61[19.64–28.05] mg/kg per day, P = 0.24) did not differ significantly.
Correlations
Within the patient group, VLDL apoB ASR was significantly correlated with plasma triglyceride concentration (P = 0.02, Rs = 0.59), plasma HDL cholesterol concentration (P = 0.03, Rs = -0.55), apoB plasma pool (P = 0.0006, Rs = 0.76), VLDL triglyceride concentration (P = 0.02, Rs = 0.59), VLDL apoB concentration (P = 0.001, Rs = 0.73), and VLDL triglyceride to apoB molar ratio (P = 0.02, Rs = 0.63). VLDL apoB FSR was significantly correlated with VLDL cholesterol (P = 0.01, Rs = -0.60) and plasma NEFA levels (P = 0.02, Rs = -0.60).
Discussion
In this study, we have confirmed that hypopituitarism is associated with deranged plasma lipid levels. In common with others, we have demonstrated a significantly lower HDL cholesterol (1, 2) and higher triglyceride level (1, 3, 4, 5), with similar plasma NEFA levels. In the patients studied, total and LDL cholesterol levels were not increased, in contrast with other studies (1, 3, 4). However, the exclusion in this study of subjects with concurrent proatherogenic problems, such as impaired glucose tolerance, may account for these differences. VLDL particles were significantly triglyceride enriched, as assessed by an increase in the molar ratios of VLDL triglyceride to apoB. No significant differences in VLDL apoB100 FSR nor pool size were found. VLDL apoB ASR tended to be elevated, although this was not significant
Higher plasma triglyceride concentrations and triglyceride enrichment of VLDL may result from an increase in production, reduced catabolism of VLDL triglyceride, or both. Increases in hepatocyte triglyceride content are usually accommodated by an increase in VLDL particle size. When this compensatory mechanism is saturated, an increase in VLDL particle secretion is observed. The significant relationship between VLDL apoB ASR and VLDL triglyceride: The apoB ratio would support this mechanism and could explain the observed triglyceride enrichment of VLDL, with a nonsignificant increase in ASR. The reason for possible hepatocyte triglyceride excess is unclear; certainly plasma NEFA levels were not elevated in our patients, although it is conceivable that portal levels are higher. Alternatively, triglyceride may be recycled from other lipoproteins, such as HDL, but we have no evidence for this in hypopituitarism.
Reduced catabolism of VLDL triglyceride may also result in triglyceride enrichment. Triglyceride is usually removed from VLDL via two routes; the first is via lipolysis catalyzed by lipoprotein lipase. Sequential removal of triglyceride from VLDL leads to the formation of LDL. Although VLDL triglyceride was increased in this study, we did not observe an increase in LDL concentration, suggesting either a block in this pathway or a compensatory increase in LDL catabolism. Packard (19) has previously demonstrated reduced conversion of triglyceride-laden VLDL to LDL, which could explain the observed triglyceride enrichment of VLDL, with unchanged LDL levels. This, however, has not been assessed in the present study.
The second route by which VLDL triglyceride is removed is via cholesterol ester transfer protein (CETP). This enzyme, located in HDL, facilitates the transfer of cholesterol esters from HDL to VLDL in exchange for triglyceride. A reduction of CETP activity could potentially result in triglyceride-laden VLDL. Data concerning CETP activity in hypopituitary subjects are limited, although Beentjes (26) recently demonstrated a decrease in CETP activity with GH replacement in hypopituitary subjects.
The relationship between raised plasma triglyceride levels and the development of CHD was first recorded in 1972 (27) and has been confirmed in many studies since then (28, 29, 30, 31). Although LDL is considered the atherogenic lipoprotein, VLDL may also cross the vascular intima and may act in a similar way to oxidized LDL (14). In addition, hypertriglyceridemia promotes the formation of small, dense LDL, which is more susceptible to oxidative modification (32) and is strongly associated with the development of coronary heart disease (33). A large pool of triglyceride-rich VLDL may encourage the formation of small, dense LDL. Indeed, an increase in small, dense LDL has previously been demonstrated in hypopituitary subjects (7). The possibility of transfer of cholesterol esters from HDL to VLDL contributes to the low HDL levels often seen in hypertriglyceridemia, as demonstrated in this study.
GH may be important in HDL metabolism, as demonstrated by GH-induced reversal of the low HDL levels observed in hypopituitarism (34, 35). However, the hypertriglyceridemia associated with hypopituitarism has rarely been ameliorated by GH replacement (1, 3, 18), even when large numbers were considered in a multicentered study (4), suggesting an alternative etiology. Although the effect of untreated GH deficiency on lipid metabolism cannot be discounted, routine endocrine replacement may also contribute to the dyslipidemia observed. Although cortisol profiles in these patients remained within the normal range, inappropriate and nonphysiological replacement with hydrocortisone (36), thyroxine (37), and sex hormones (38, 39) have all been associated with lipid abnormalities.
Footnotes
This work was supported in part by Pharmacia-Upjohn Pharmaceuticals Ltd. and the Child Growth Foundation.
Abbreviations: -KIC, -Ketoisocaproic acid; APE, atom per
excess; apoB, apolipoprotein B; ASR, absolute synthetic rate; BMI, body
mass index; CETP, cholesterol ester transfer protein; FSR, fractional
synthetic rate; HDL, high-density lipoprotein; LDL, low-density
lipoprotein; NEFA, nonesterified fatty acid; Rs, Spearman’s rank;
VLDL, very low-density lipoprotein.
Received January 31, 2001.
Accepted April 27, 2001.
References
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