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Original Studies |
Departments of Clinical and Experimental Medicine and Internal Medicine, University of Padova, 35100 Padova, Italy
Address all correspondence and requests for reprints to: Angelo Avogaro, M.D., Cattedra di Malattie del Metabolismo, Via Giustiniani 2, 35100 Padova, Italy. E-mail: avogaro{at}ux1.unipd.it
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Abstract |
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Fourteen normal volunteers were studied under baseline conditions and then randomly allocated to two of the following four studies: 1) long chain triglyceride (LCT) emulsion and heparin infusion (n = 7), 2) infusion of an emulsion containing 56% medium chain triglycerides (MCT) and 44% LCT plus heparin (n = 7), 3) infusion of LCT and heparin preceded by an iv bolus of 900 mg lysine-salycilate (ASA; n = 7), and 4) after an iv bolus of ASA (n = 7). Basal forearm blood flow (FBF), endothelium-dependent vasodilation in response to intraarterial acetylcholine (Ach), and endothelium-independent vasodilation in response to intraarterial nitroprusside were assessed by venous occlusion plethysmography. Both LCT and MCT infusions significantly increased basal FBF from 1.58 ± 0.35 to 2.60 ± 0.76 and 2.28 ± 0.56 mL/min·100 mL tissue, respectively (both P < 0.05). This increase was also observed for LCT plus heparin, but not after ASA alone. The percent increase in FBF during Ach was lowered during both LCT (252 ± 34% of the ratio infused/control arm at maximal Ach dose) and MCT (255 ± 41%) compared to the baseline conditions (436 ± 44%; both P < 0.05). The response to Ach was also lower during LCT plus ASA, whereas it was similar to baseline with ASA alone. No differences were observed in the response to nitroprusside among the experimental conditions.
In conclusion, 1) the effect of FFA on endothelium-dependent vasodilation is independent of their chain length; 2) both LCT and MCT increase baseline FBF, independently from cyclooxygenase inhibition; and 3) acute ASA administration does not affect endothelium-dependent vasodilation. The FFA effect on the endothelial response to Ach may contribute to altered endothelial function and, hence, to the development and progression of atherosclerotic cardiovascular disease.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Conditions of insulin resistance are characterized by inappropriate elevation of plasma free fatty acids (FFA) (7). The increase in the circulating levels of these substrates may affect insulin action by impairing the intrinsic ability of insulin to promote glucose utilization (expressed by the artero-venous glucose difference), either by competition with glucose at the level of the oxidative pathway (8) or by affecting glucose transport and phosphorylation (9). Experimental data exist to show that oleic acid inhibits the constitutive NO synthase (NOS III) in cultured bovine pulmonary artery endothelial cells (10). These findings lend support to the hypothesis that increased FFA availability might affect insulin-mediated vasodilation, as initially observed in man by Steinberg et al. (11). We reported that in conditions associated with marked plasma FFA concentration, as occurs after insulin withdrawal in type 1 diabetic subjects, a significant alteration in NO-mediated vasodilation develops (12). Thus, the possibility arises that FFA may induce insulin resistance acting at the levels of both insulin-mediated glucose metabolism and insulin-mediated vasodilation.
However, several aspects of these possible mechanisms remain to be clarified, as circulating FFA are part of a family of acids of different carbon atom chain length and composition. For instance, although oleic [18:1(cis)] and linoleic [18:2(cis)] acids inhibit NOS III, stearic acid (18:0) does not (13). Moreover, FFA such as linoleic acid are precursors of arachidonic acid and PGs (14), i.e. potent vasoactive agents.
Therefore, the objectives of the present investigation were 1) to define the role of different chain lengths on endothelium-dependent vasodilation, and 2) to verify the possible implication of PGs in the vascular response to elevation of plasma FFA levels. These aims may be relevant not only because they provide insights into the metabolic regulation of endothelial modulation, but also for clinical reasons. For instance, commercially available lipid emulsions can cause cardiovascular side-effects (15). These negative effects may be explained by different compositions in terms of triglyceride chain length. Moreover, the higher ketogenic potential of medium chain triglycerides may affect endothelium-dependent vasodilation (16).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 14 normal volunteers were studied. Their clinical
features are shown in Table 1
. All of
them had normal plasma concentrations of circulating lipids and normal
urinary albumin excretion rates. A full medical history was recorded,
and physical examination was performed to exclude known hepatic, renal,
or myocardial dysfunction or other endocrine diseases. Moreover, to
rule out preexisting factors known to affect endothelial function, only
subjects with no history of hypertension and/or hyperlipidemia were
recruited. All subjects followed an isocaloric diet, with three meals a
day (50% carbohydrate, 35% fat, and 15% protein) for at least 30
days before the study. Nitrate-rich food was prohibited for at least
24 h before the study. Informed, written consent was obtained from
each patient after the purpose, nature, and potential risks of the
study were explained. The study protocol was approved by the ethical
committee of the School of Medicine, University of Padova.
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Subjects were randomly allocated to two of four different protocols. Metabolic and hemodynamic evaluations were carried out in the morning after a 10- to 12-h overnight fast; they were performed on different days, 2–3 weeks apart. Women were studied during the follicular period of their menstrual cycle. Upon admission to our Metabolic Unit, both a brachial artery and two superficial veins of the contralateral arm were cannulated, and an arterial blood sample was collected for the determination of basal plasma levels of FFA, glucose, insulin, and blood ketone body concentrations. After completion of baseline blood flow determination (see below), four different studies were carried out. Studies 1 and 2 were randomly performed before studies 3 and 4 were carried out.
Study 1. Endothelium-dependent and independent vasodilatory
responses were randomly assessed before and after 120-min continuous iv
infusion of long chain triglycerides (LCT; 10% Intralipid; see
composition in Table 2; 2 mL/min) plus
heparin (40 U/kg·h) as previously reported (17).
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; 2 mL/min) plus heparin (40
U/kg·h). Study 3. Study 3 was identical to study 1, with the exception that the LCT/heparin infusion was preceded by an iv bolus of lysine-salycilate (ASA; 900 mg) (18).
Study 4. Endothelium-dependent and independent vasodilatory responses were randomly assessed after iv ASA administration 2 h before the assessment of endothelium-dependent and independent vasodilatory responses. No lipid emulsion was infused on this occasion.
Before the infusion of each test substance, normal saline was administered. During this saline infusion, both acetylcholine (Ach) and sodium nitroprusside (SNP; see below) were intraarterially administered for estimation of baseline endothelial function.
Assessment of endothelial function
On the morning of each study an 18-gauge catheter was inserted under local anesthesia (1% xylocaine) and sterile conditions into the brachial artery of the nondominant arm of each subject for the regional infusion of vasoactive agents. The forearm was allowed to rest comfortably on supports, slightly above the heart level. The arterial line was kept patent by a normal saline infusion (0.3 mL/min). Two superficial veins of the contralateral arm were then cannulated: the first venous access was used for the infusion of all nonvasoactive test substances, and the second one was use for venous blood sampling.
After allowing the subject to become acquainted with the experimental environment (20–30 min), basal forearm blood flow (FBF) was assessed by venous occlusion plethysmography (Angioflow 2, Microlab Elettronica, Padova, Italy). The hand was excluded from the circulation by inflating a wrist cuff to 200 mm Hg for 30 s before and during each blood flow measurement. Each FBF determination represented the average value of 10 separate measurements performed at 12-s intervals. Intraindividual variability of resting blood flow was estimated by 10 consecutive readings. Under these conditions, the coefficient of variation ranged from 2–5% for FBF and from 1–2% for blood pressure (19, 20). Blood pressure was recorded immediately before each measurement with a noninvasive technique (Finapress, Ohmeda, Englewood, CA). The endothelium-dependent and independent dilatations were then assessed in a random order by sequential intrabrachial infusion of Ach and SNP, respectively. Endothelium-dependent vasodilation was assessed by determination of FBF in response to a stepwise Ach infusion (7.5, 15, and 30 µg/min). Ach solution was made up in normal saline, and each dose was delivered for 5 min at a rate of 0.4 mL/min. Endothelium-independent vasodilation was assessed as the response of the FBF to SNP infusion (0.3, 3, and 10 µg/min at a rate of 0.4 mL/min, each dose for 5 min). Blood flow was continuously recorded during each infusion step starting 1 min after the beginning of each dose. The second stimulation (Ach or SNP, according to randomization) was performed 45 min after the completion of the initial evaluation, when blood flow parameters had returned to baseline conditions. Endothelium- and nonendothelium-dependent vasodilations were measured before (the baseline study) and after administration of lipid emulsion or ASA as described above; the first challenge started 45 min after beginning of each lipid infusion.
Blood flow measurements were performed in the contralateral (control) forearm as well, to ensure that systemic effects did not occur during the intraarterial infusion of vasoactive agents. Therefore, the ratio of forearm blood flow (infused/control arm) measured in response to drugs was expressed as a percentage of the ratio measured during the control period.
Biochemical assays
Plasma glucose was measured with a glucose oxidase method on a Glucose Analyzer 2 (Beckman Coulter, Inc., Fullerton, CA). Blood 3-hydroxybutyrate (3-BOH) (21) and acetoacetate (AcAc) concentrations were measured by fluorometric technique (22). Plasma FFA concentrations were determined using a microenzymatic technique (23). Plasma insulin concentrations were measured by standard RIA procedures (24).
Calculations and statistical analysis
Results are presented as the mean ± SD. Mean blood pressure was calculated as diastolic blood pressure plus one third of the difference between systolic and diastolic blood pressure. Statistical analyses of the dose-response curves for Ach and SNP were performed for the changes from baseline to account for baseline variability. Two-way ANOVA for repeated measures was performed to compare FBF dose-response curves among studies followed by the Fischer least significant difference test. Comparisons between groups were performed using unpaired Student’s t test for normally distributed data and the Mann-Whitney test when the normality test failed. Statistical significance was considered for P < 0.05 for two-tailed analysis.
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Results |
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)No differences were observed in the fasting plasma glucose concentration in the morning of each study. In response to the combined lipid emulsion/heparin infusion, there was a 10-fold increase in plasma FFA concentration with or without the ASA bolus. ASA alone had no effect on plasma FFA levels. The blood total ketone body (AcAc plus 3-BOH) concentration increased during lipid emulsion (P < 0.05 vs. baseline), with a greater increase during MCT than LCT infusion (P < 0.05). ASA alone had no effect on the blood levels of these substrates. The 3-BOH/AcAc ratio significantly increased during lipid emulsion infusion. Again, the ratio was higher during MCT than LCT.
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, during the infusion of intervention substances we
did not observe any significant response in plasma glucose
concentrations, although the baseline plasma insulin concentration
increased whenever lipid emulsions were administered. Endothelium-dependent and independent vasodilation
Baseline parameters (Table 4).
There was no difference in the baseline FBF. However, both LCT
(2.60 ± 0.76 mL/min·100 mL tissue) and MCT (2.28 ± 0.56
mL/min·100 mL tissue) infusions were associated with a significant
(P < 0.05 vs. baseline) increase in FBF.
The same increase was observed when the ASA administration preceded
lipid infusion (2.75 ± 0.65 mL/min·100 mL tissue;
P < 0.05 vs. baseline), wherea baseline FBF
was not affected by the isolated ASA injection.
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SNP and Ach infusions. No differences were observed in response to SNP among the experimental conditions. During the highest dose of SNP (10 µg/min), FBF increased by 495 ± 88% in the baseline period, by 485 ± 61% during LCT, by 501 ± 41% during MCT, by 499 ± 47% during LCT plus ASA, and by 511 ± 36% during ASA alone, respectively. These SNP-stimulated FBF values were all significantly higher (P < 0.0001) than those observed in the nonstimulated period or those observed at lower SNP doses (P < 0.01).
On the contrary, a different behavior was observed in response to Ach.
Percentagewise, the increase in FBF during Ach was smaller during both
LCT (252 ± 34% of the ratio infused/control arm at maximal Ach
dose; P < 0.001) and LCT (255 ± 41%;
P < 0.001) compared to what was found in the baseline
period (436 ± 44%). The same response to Ach was recorded after
LCT plus ASA (261 ± 50%; P < 0.001
vs. baseline), whereas it was similar to baseline with ASA
alone. These responses are graphically depicted for each subject as
individual dose-response curves for each study in Fig. 1.
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, the
absolute increases in FBF during Ach were significantly lower during
LCT, MCT, and LCT plus ASA compared to those observed in the baseline
condition and during ASA alone (both P < 0.01 or
less). It should be noted, however, that the absolute maximal response
was similar in all the experimental conditions (LCT, 6.55 ± 0.42;
MCT, 5.81 ± 0.84; LCT plus ASA, 6.62 ± 0.68; ASA, 6.83
± 0.36 mL/min·100 mL tissue).
Systemic mean blood pressure as well as heart rate did not change
during the intraarterial Ach infusion in any experimental condition
(Table 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism(s) responsible for the increase in FBF is not clear, as the infusion of lipid emulsion has been shown to induce a permissive action to adrenergic compounds (25), and accumulation of vaso-constricting PGs (26); this would imply a decreased rather than increased blood flow at constant pressure values. From our data it could be speculated that this FFA-induced increase in FBF is not mediated by PG-sensitive mechanisms, because the same degree of FFA-mediated increase in FBF was observed even after ASA administration. It is of note that during lipid infusion a 50% increase in the plasma insulin concentration occurred. This rise may reflect either a direct stimulation of FFA on the ß-cell (27) or a compensatory hypersecretion due to a state of insulin resistance induced by these substrates (28). This rise in the plasma insulin concentration may have triggered vasodilation directly through endothelial NO release (29); alternatively, lipid infusions may have reduced the sympathetic nervous activity and induced a simultaneous increase in parasympathetic to sympathetic nerve activity ratio (30). Another possibility is the change in the intracellular redox state and the increase in blood ketone body concentration induced by accelerated FFA metabolism. The shift in the intracellular redox state may lead to increased intraorgan blood flow and vascular dysfunction (31). We recently found that ketone bodies, the product of FFA oxidation, may affect vascular function in insulin-withdrawn type 1 diabetic patients (12). However, we are inclined to rule out a significant effect of blood ketones in the present experimental setting. MCT infusion exerts a greater ketogenic effect than LCT (16), as also found in the present study. Despite these differences, the increment in baseline FBF was the same with MCT and LCT infusions.
Eventually, we found that ASA alone had no significant effect on endothelial function. This result is at variance with previous report showing that acute intraarterial administration of ASA was capable of decreasing FBF in a dose-dependent manner as a consequence of the inhibition of the synthesis of vasodilating PGs (32). This conclusion, however, is challenged not only by our results but also by other studies (33).
An apparently different picture developed when the endothelium-dependent vascular response was ascertained. The percent increase in FBF during Ach was smaller during both LCT and MCT compared to that found under basal conditions. These data, therefore, seem to confirm previous findings suggesting that although increased levels of FFA do not influence endothelium-independent vasodilation, they impair endothelium-dependent vasodilation (2). As previously observed for the basal FBF changes, there was no specific chain length effect, as both MCT and LCT elicited the same response. These negative effects of increased plasma FFA levels on Ach-mediated vasodilation were evident not only on a percentage basis, but also in terms of absolute changes from baseline.
However, when the maximal FBF attained in each experimental condition is considered, the lack of any significant difference must be acknowledged. Whether these values represent maximal vasodilation or express the inability to evoke a normal vasodilatory response above the baseline condition cannot be defined by the present experimental procedure. Nonetheless, when basal FBF between groups differs, the interpretation of the physiological response may be problematic; as outlined by Benjamin et al. observations in the same subjects studied on different occasions suggest that the absolute response to vasodilator compounds usually increases with increasing basal flow (34). If that is the case, the FFA-induced impairment of endothelial function should have been underestimated in our study, lending further support to the role of these compounds in the vasodilatory modulation. It has been claimed that an elevated plasma FFA concentration may induce insulin resistance by direct metabolic competition (28) as well as by hampering the increase in FBF (11). Our results suggest that the contribution of the latter mechanism must be trivial, as a 10-fold elevation in plasma FFA levels did not affect the absolute maximal FBF attained in response to a muscarinic, endothelium-dependent, agent. Therefore, the reduction in glucose utilization that accompanies the elevation of plasma FFA concentration must be largely accounted for by intracellular effects of these substrates.
It has been previously shown that an impaired vasodilatory response to Ach may result from high serum triacylglycerol levels (35), although their postprandial, rather their fasting, plasma levels interfere with endothelial-mediated vasodilation (36). Both LCT and MCT infusions generally increase triacylglycerols by 3-fold (37). Therefore, at least part of the effect of lipid emulsion infusions on the endothelium-dependent vasodilatory response could be due to increased concentrations of these substrates in plasma. In this study we do not report the triglyceride concentrations before and during lipid emulsion infusion, but our data appear consistent with those of Vogel and colleagues, who showed that a high fat diet is associated with a blunted flow-mediated vasodilation, a sensitive NO-dependent index of endothelial function (38, 39). However, in vitro findings demonstrate that triolein and tripalmitin have a vasorelaxant, rather than a vasoconstrictor, effect (40, 41). Thus, the relationship between increased FFA and increased triacylglycerol levels on endothelial function will need further investigations.
No specific assay for the circulating levels of series 2 PGs was
carried out in the present study, but it is known that linoleic and
-linolenic acid can affect PG synthesis (42). The blunted percent
response to Ach during the infusions of lipid emulsions may be due to
the concomitant synthesis of vasoconstricting PGs, such as
PGH2. In our study lysine salycilate was employed
to inhibit PG synthesis. However, no difference was detected between
studies with and without salycilate administration, suggesting that, at
least under the present experimental condition, the inhibition of PG
pathway contribute little to the change in FBF observed in response to
lipid infusion. These findings appear to be similar to those of Duffy
et al., who showed that aspirin administration did not
affect the responses to Ach or nitroprusside (32), and would exclude an
enhanced release of NO after prostacyclin inhibition (43).
Our data may have clinical implications. Lipid emulsions are frequently employed for nutritional purposes, and triglycerides esterified with fatty acids with a chain longer than 16 carbon atoms are commonly used. These emulsions contain not only a greater proportion of long chain fatty acids than those with MCT, but also a greater proportion of linoleic acid, which is a natural precursor of the series 2 PGs (14). From the point of view of possible interference with endothelial function, our results now provide evidence that no major difference occurs between the two preparations. The quite similar responses obtained with LCT and MCT infusions are in disagreement with the marked increase in systemic vascular resistance observed in dogs infused with MCT emulsion by Van de Velde et al. (15). These effects are the likely consequence of the octanoate present in the MCT emulsions, which is known to affect Na+/K+-adenosine triphosphatase activity (44). However, it should be pointed out that the rate of MCT infusion in those animals was almost 5 times higher than that used in this study, a difference that may well explain the discrepancy between their and our findings.
Finally, it must be acknowledged that we coinfused lipid emulsions with heparin to enhance triglyceride breakdown. It has been recently shown that heparin may decrease the production of endothelial derived NO in rats (45), and that at least in hypertensive patients, this compound may show a blood pressure-lowering effect due to a NO-mediated suppression of endothelin-1 (46). Therefore, a vasodilatory effect of heparin could have been masked by the coinfusion of lipid emulsion. However, our data tend to exclude an interfering effect of heparin, because, although we observed an increased baseline blood flow during the coinfusion of lipid emulsion and heparin, endothelium-dependent vasodilation was reduced upon Ach challenge.
In conclusion, we have shown that both LCT and MCT emulsions increase baseline FBF independently from PG synthesis, but they blunt the ability of endothelium-derived NO to cause a normal vasodilatory increment above baseline. These findings can be interpreted to suggest that an elevated plasma FFA concentration may favor the development and progression of atherosclerotic cardiovascular disease throughout an abnormal regulation of endothelium-dependent vasodilation. Moreover, our results lend support for a primary intracellular, metabolic effect contributing to the impairment of glucose metabolism associated with increased plasma FFA availability.
Received May 17, 1999.
Revised September 21, 1999.
Accepted October 27, 1999.
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-adrenergic sensitivity. Hypertension. 25:774–778.This article has been cited by other articles:
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  M. D. Jensen Adipose tissue as an endocrine organ: implications of its distribution on free fatty acid metabolism Eur. Heart J. Suppl., May 1, 2006; 8(suppl_B): B13 - B19. [Abstract] [Full Text] [PDF]
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 R. E. Maser and M. J. Lenhard Cardiovascular Autonomic Neuropathy Due to Diabetes Mellitus: Clinical Manifestations, Consequences, and Treatment J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5896 - 5903. [Abstract] [Full Text] [PDF]
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 M. Srinivasan, P. Herrero, J. B. McGill, J. Bennik, B. Heere, D. Lesniak, V. G. Davila-Roman, and R. J. Gropler The Effects of Plasma Insulin and Glucose on Myocardial Blood Flow in Patients With Type 1 Diabetes Mellitus J. Am. Coll. Cardiol., July 5, 2005; 46(1): 42 - 48. [Abstract] [Full Text] [PDF]
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J. Pleiner, G. Schaller, F. Mittermayer, M. Bayerle-Eder, M. Roden, and M. Wolzt FFA-Induced Endothelial Dysfunction Can Be Corrected by Vitamin C J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2913 - 2917. [Abstract] [Full Text] [PDF]
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) in FBF (milliliters
per min/100 mL tissue) in response to graded intrabrachial artery
infusion of acetylcholine (Ach) and during infusion of saline, LCT,
MCT, LCT plus ASA, and ASA alone