March 2, 2011 -- Tennis star Serena Williams is recovering from a pulmonary embolism and a hematoma resulting from her treatment, according to media reports.
What is a pulmonary embolism? How can something so scary happen to a world-class athlete? Do people fully recover from a pulmonary embolism?
To answer these and other questions, WebMD consulted Shirin Shafazand, MD, MPH, assistant professor of medicine in the division of pulmonary critical care at the University of Miami Miller School of Medicine.
Shafazand has not examined Williams and has not seen her medical records. She commented on publicly available details of Williams' condition and on her extensive experience treating patients with pulmonary embolisms.
Shortly after winning her fourth Wimbledon title last July, Williams cut her foot on a shard of glass. The severe cut required surgery and 18 stitches. Although she played an exhibition match shortly thereafter, continuing problems with the foot has kept her out of competition since then.
According to a statement from her representative in People magazine, Williams was in New York last week undergoing further treatment for her foot injury. She flew back to Los Angeles and apparently suffered a pulmonary embolism during or shortly after the flight.
On Feb. 28, she "underwent emergency treatment" for a hematoma she suffered as a result of her treatment for pulmonary embolism.
Williams is reported to be recovering.
A pulmonary embolism is a blood clot that blocks a major artery feeding the lungs.
These clots usually arise in the leg, usually in a deep vein. Doctors call such a clot a thrombosis. A clot arising in a deep leg vein is called a deep venous thrombosis or DVT. DVTs often arise after a period of inactivity and are particularly common after long airplane flights. A clot originating in a deep leg vein in some cases will break free and travel to the lungs, causing a pulmonary embolism.
Williams may have been relatively inactive because of the foot injury. The risk of DVT is increased with inactivity.
"Her injury could have led to a clot in the leg," Shafazand suggests. "And New York to California is quite a long trip -- and that could increase risk of the slowing down of blood flow in the leg, which could lead to a DVT."
Some people with very small pulmonary embolisms never notice them. But larger clots block blood flow to significant portions of the lungs.
"A large pulmonary embolism cuts blood circulation to the lungs and decreases oxygen levels in the body. A patient can very quickly deteriorate and die," Shafazand says. "And the heart, which is supposed to pump the blood through the arteries, can fail because it cannot stand the pressure buildup from the blockage."
【关键词】 Role
Voltage-gated potassium (KV) channels play an essential role in regulating pulmonary artery function, and they underpin the phenomenon of hypoxic pulmonary vasoconstriction. Pulmonary hypertension is characterized by inappropriate vasoconstriction, vascular remodeling, and dysfunctional KV channels. In the current study, we aimed to elucidate the role of PKC and its adaptor protein p62 in the modulation of KV channels. We report that the thromboxane A2 analog 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2 methyl acetate (U46619[GenBank]) inhibited KV currents in isolated mice pulmonary artery myocytes and the KV current carried by human cloned KV1.5 channels expressed in Ltk– cells. Using protein kinase C (PKC)–/– and p62–/– mice, we demonstrate that these two proteins are involved in the KV channel inhibition. PKC coimmunoprecipitated with KV1.5, and this interaction was markedly reduced in p62–/– mice. Pulmonary arteries from PKC–/– mice also showed a diminished [Ca2+]i and contractile response, whereas genetic inactivation of p62–/– resulted in an absent [Ca2+]i response, but it preserved contractile response to U46619.[GenBank] These data demonstrate that PKC and its adaptor protein p62 play a key role in the modulation of KV channel function in pulmonary arteries. These observations identify PKC and/or p62 as potential therapeutic targets for the treatment of pulmonary hypertension.
Voltage-gated potassium (KV) channels play an essential role in regulating vascular smooth muscle function. They make a substantial contribution to whole-cell K+ conductance and resting membrane potential in pulmonary artery smooth muscle cells (PASMCs), and its inhibition causes membrane depolarization, activation of L-type Ca2+ channels (CaL), increases in [Ca2+]i, and vasoconstriction (Barnes and Liu, 1995; Archer et al., 1998; Yuan et al., 1998b). These channels are common targets of pulmonary vasoconstrictor stimuli such as hypoxia, thromboxane A2 (TXA2), 5-hydroxytryptamine, endothelin-1 or angiotensin-II (Archer et al., 1998; Shimoda et al., 2001; Cogolludo et al., 2003, 2006). In addition, decreased expression or function of KV channels in PASMCs has been involved in the pathogenesis of pulmonary arterial hypertension (PH) (Weir et al., 1996, Yuan et al., 1998a; Pozeg et al., 2003). From the variety of KV channels expressed in PASMC (Platoshyn et al., 2006), special interest has been paid to KV1.5, because decreased expression or activity and mutations of KV1.5 occur in human (Yuan et al., 1998a; Remillard et al., 2007) and experimental (Archer et al., 1998; Pozeg et al., 2003) idiopathic and hypoxic PH, and in vivo gene transfer of KV1.5 reduces PH (Pozeg et al., 2003).
TXA2 is a prostanoid synthesized by cyclooxygenase with potent vasoconstrictor, mitogenic, and platelet aggregant properties via activation of thromboxane-endoperoxide (TP) receptors (Halushka et al., 1989). The vasoconstrictor effects of TXA2 are particularly pronounced in the pulmonary vascular bed, where it participates in the control of vessel tone under physiological and pathological situations, including PH. We have previously reported that in intact PAs and freshly isolated PASMCs, TXA2, via activation of TP receptors, inhibits KV channels, leading to membrane depolarization, activation of L-type Ca2+ channels, and vasoconstriction. Furthermore, using a protein kinase C (PKC) pseudosubstrate inhibitory peptide (PKC-PI), we provided evidence for the role of this kinase as a link between TP receptor activation and KV channel inhibition (Cogolludo et al., 2003, 2005). PKC (together with PKC/) belongs to the atypical PKC (aPKC) subclass. Both aPKCs play key roles in different signaling pathways regulating cell growth, survival, and differentiation (Moscat and Díaz-Meco, 2000). The aPKCs share with other members of their family a conserved catalytic domain, but they display a clearly distinct regulatory region because they have been shown to be independent of Ca2+, diacylglycerol, and phorbol esters, all of which are potent activators of other PKC isoforms. PKC is activated by phosphatidylinositols, arachidonic acid, and other lipids (Hirai and Chida, 2003) as well as by a variety of mediators, including insulin (Liu et al., 2006), thromboxane A2 (Shizukuda and Buttrick, 2002; Cogolludo et al., 2003, 2005), angiotensin II (Gayral et al., 2006; Godeny and Sayeski, 2006), or proinflammatory cytokines (Frey et al., 2006).
The mechanism underlying the activation of aPKCs responsible for its diverse physiological functions remains unclear, but several groups have identified a number of aPKC-interacting proteins, including p62 (also called ZIP1 or sequestosome 1), Par-4, Par-6, and MEK5 (Moscat and Diaz-Meco, 2000). It is noteworthy that nerve growth factor and catecholamines have been reported to increase the expression of p62, enabling the formation of the PKC-p62-KV complex, which results in a hyperpolarizing shift in the KV current activation curve (Gong et al., 1999; Kim et al., 2004, 2005).
The role of PKC on pulmonary vasoconstriction has been widely reported (Ward et al., 2004); however, many of these studies have been conducted with PKC modulators of dubious selectivity, thereby limiting their conclusions. Molecular biology and genetic approaches and the currently available isoform-selective PKC inhibitors have made possible the elucidation of the involvement of specific PKC isoforms in cellular processes (such as vascular contractility) (Salamanca and Khalil, 2005). However, recent evidence suggests that some considered isoform-specific PKC inhibitors, such as myristoylated PKC pseudosubstrate peptide, may exert other effects unrelated to inhibition of PKC; thus, they should be used with caution (Krotova et al., 2006).
Therefore, in the present study, we aimed to further characterize the signaling pathway modulating KV currents in PAs. Using PKC–/– and p62–/– mice, we provide evidence for the interaction of PKC with KV channels, which further support the role of this interaction in TXA2-induced effects. In addition, we hypothesized that the PKC-KV-L-type Ca2+ channels pathway might involve other proteins such as p62. This possibility was tested by analyzing the modulation of KV channels in wild-type and p62 homozygous null mice.
All experiments were carried out in accordance with the European Animals Act 1986 (Scientific Procedures), and they were approved by our institutional review board.
Animals. Lungs from PKC–/– (mixed C57BL/6 and SV129J background), p62–/– (C57BL/6), and corresponding wild-type mice (6–8 weeks old; either sex) were generously supplied by Drs. J. Moscat and M. T. Diaz-Meco (both from the Genome Research Institute, University of Cincinnati, Cincinnati, OH). These mice were generated as described previously (Leitges et al., 2001; Duran et al., 2004). PAs from male Wistar rats (250–300 g) were also used in these experiments.
Tissue Preparation and Cell Isolation. Second-order branches of the PA (internal diameter, 0.5 mm) isolated from mice were dissected into a nominally calcium-free physiological salt solution (PSS) of the following composition: 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.3, with NaOH. Endothelium denuded PAs were cut into small segments (2 x 2 mm), and cells were isolated in Ca2+-free PSS containing 1 mg/ml papain, 0.8 mg/ml dithiothreitol, and 0.7 mg/ml albumin. Cells were stored in Ca2+-free PSS (4°C) and used within 8 h of isolation.
Electrophysiological Studies. Membrane currents were measured using the whole-cell configuration of the patch-clamp technique (Cogolludo et al., 2003) normalized for cell capacitance and expressed in picoamperes per picofarad. Membrane potential (Em) was measured under current-clamp configuration. KV currents were recorded under essentially Ca2+-free conditions using an external Ca2+-free PSS and a Ca2+-free pipette (internal) solution (see Solutions and Chemicals). Ltk– cells stably expressing hKV1.5 channels (Valenzuela et al., 1995) were superfused with PSS containing 1 mM CaCl2. Currents were evoked after the application of 200-ms depolarizing pulses from –60 mV to test potentials from –60 to +40 mV in 10-mV increments. All experiments were performed at room temperature (22–24°C).
[Ca2+]i Recording. PA rings were incubated for 80 min at room temperature in Krebs' solution containing the fluorescent dye fura-2 acetoxymethyl ester (5 x 10–6 M) and 0.05% cremophor EL, and then they were mounted in a fluorimeter (model CAF 110; Jasco, Tokyo, Japan). PA rings were alternatively illuminated (128 Hz) with two excitation wavelengths (340 and 380 nm), and the emitted fluorescence was filtered at 505 nm (Pérez-Vizcaíno et al., 1999). The ratio of emitted fluorescence (F340/F380) obtained at the two excitation wavelengths was used as an indicator of [Ca2+]i. Arteries were stimulated with 30 and 300 nM U46619[GenBank], added in a cumulative manner. In preliminary experiments in wild-type mice, these concentrations produced 60 and 80% of the maximal response, respectively. The [Ca2+]i signal in each vessel was calibrated according to the Grynkiewicz equation by sequential addition of 15 µM ionomycin and 10 mM EGTA at the end of the experiment.
Coimmunoprecipitation and Western Blot Analysis. Mice lungs were rapidly frozen in liquid nitrogen. In some experiments, rat PA were placed in warm Krebs' solution and then in the absence or presence of 1 µM U46619[GenBank] for 30 s and then rapidly frozen. Frozen tissues were homogenized in a glass potter in 200 µl of a buffer of the following composition: 10 mM HEPES, pH 8, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 6 µM aprotinin, 9 µM leupeptin, 11 µM N-p-tosyl-L-lysine chloromethyl ketone, 5 mM NaF, 10 mM Na2MoO4, 1 mM NaVO4, 0.5 mM phenylmethanesulfonyl fluoride, and 10 nM okadaic acid. Homogenates were centrifuged at 13,000g for 5 min at 4°C, and the supernatant fraction was collected. For immunoprecipitation, 60 µg of protein was incubated for 2 h with anti-PKC or anti-KV1.5 antibody at 4°C, followed by the addition of protein A/G beads and further incubation overnight. These immune complexes or 20 µg of the homogenates from mice lungs or rat PA were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane for Western blotting as described previously (Cogolludo et al., 2003). Membranes were probed for KV1.5-, PKC-, and p62-like immunoreactivity.
Solutions and Chemicals. For the single cell electrophysiological studies, the composition of the Ca2+-free bath solution (external Ca2+-free PSS) was as follows: 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, buffered to pH 7.3 with NaOH. The Ca2+-free pipette (internal) solution contained 110 mM KCl, 1.2 mM MgCl2, 5 mM Na2ATP, 10 mM HEPES, and 10 mM EGTA, pH adjusted to 7.3 with KOH. The Krebs' solution used for tissue bath experiments included 118 mM NaCl, 4.75 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 2.0 mM CaCl2, 1.2 mM KH2PO4, and 11 mM glucose. This solution was gassed with a 95% O2, 5% CO2 mixture at 37°C. U46619[GenBank] was obtained from Sigma Chemical Co. (Tres Cantos, Spain). [1S-1,2,5]-[5-Methyl-2-(1-methylethyl) cyclohexyl] diphenyl phosphine oxide (DPO-1) was from Tocris Cookson Inc. (Bristol, UK), secondary horseradish peroxidase-conjugated antibodies and fura-2 acetoxymethyl ester were from Calbiochem (Barcelona, Spain), rabbit anti-KV1.5 was from Alomone Labs (Jerusalem, Israel), goat anti-PKC was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and guinea pig anti-p62 was from Progen (Heidelberg, Germany).
Statistical Analysis. Data are expressed as means ± S.E.M.; n indicates the number of arteries or cells tested. All experiments were conducted in arteries or cells from at least four different animals. Statistical analysis was performed using Student's t test for paired or unpaired observations. Differences were considered statistically significant when p was less than 0.05.
Role of PKC in KV Current Inhibition Induced by TXA2. A family of KV currents [IK(V)] were obtained in mice PASMCs when eliciting depolarizing steps from –60 to +40 mV (Fig. 1, A and B) from a holding potential of –60 mV. The magnitude of the currents, the threshold voltage for activation, and the current-voltage relationship (Fig. 1, C and D) was similar in PASMCs from wild-type and PKC–/– mice (e.g., current density at +40 mV was 9.1 ± 1.9 and 8.7 ± 0.8 pA/pF, respectively). Current inactivation was also similar in both strains (i.e., at 200 ms, the current decayed by 11.5 ± 3 and 12.1 ± 3.8%, respectively). Currents were recorded before (control) and after addition of the TXA2 analog U46619.[GenBank] U46619 (100 nM) caused a significant inhibition of KV currents in the whole range of channel activation in PASMCs from wild-type mice (Fig. 1A). The degree of current inactivation at +40 mV was increased by U46619[GenBank] (i.e., at 200 ms, the current decayed by 25.6 ± 4.8%; p < 0.05). In addition, U46619[GenBank] induced membrane depolarization in wild-type PASMCs (Fig. 1E). However, U46619[GenBank] had no effect on either KV currents or membrane potentials in PASMC from PKC–/– mice (Fig. 1, B, D, and F).
Fig. 1. TP receptor activation leads to KV current inhibition and depolarization in PASMCs from PKC+/+ (A, C, and E) but not from PKC–/– (B, D, and F) mice. A and B, current traces for 200-ms depolarization pulses from –60 to +40 mV (in 10-mV increments) from a holding potential of –60 mV before (control) and after application of the TXA2 analog U46619 (100 nM). C and D, current-voltage relationship measured at the end of the 200-ms pulse (means ± S.E.M. of five cells). E and F, effects of 100 nM U46619 on membrane potential recorded under current clamp conditions. *, p < 0.05 and **, p < 0.01, respectively, versus control (paired Student's t test).
Role of PKC in [Ca2+]i Increase and Contraction Induced by TXA2. Changes in [Ca2+]i and contraction induced by U46619[GenBank] were simultaneously analyzed in fura-2-loaded PAs from wild-type and from PKC–/– mice. Basal levels of [Ca2+]i in PKC–/– (203 ± 40 nM; n = 6) were not significantly different from those in wild-type mice (160 ± 40 nM; n = 6). Stimulation of endothelium-denuded PA rings with 30 and 300 nM U46619[GenBank] induced a sustained elevation in [Ca2+]i and a contractile response in PAs from wild-type and PKC–/– animals (Fig. 2, A and B). However, the increase in [Ca2+]i (Fig. 2C) and the contractile response (Fig. 2D) was significantly reduced in PKC–/– mice compared with wild-type mice.
Fig. 2. PA from PKC–/– mice show reduced [Ca2+]i and contractile responses induced by TP receptor activation. A and B, simultaneous recordings of [Ca2+]i (top trace) and force (bottom trace) in PAs from PKC+/+ and PKC–/–, respectively, stimulated by 30 and 300 nM U46619. The averaged values (means ± S.E.M. of five to seven PAs) of U46619-induced increase in [Ca2+]i and force are shown in C and D, respectively. *, p < 0.05 versus PKC+/+ (unpaired Student's t test).
Role of p62 in KV Current Inhibition, [Ca2+]i Increase, and Contraction. To analyze the functional role of p62 and the PKC-p62-KV1.5 interaction, we analyzed the effects of 100 nM U46619[GenBank] on KV currents in p62–/– and the corresponding wild-type mice. The magnitude of the currents, the threshold voltage for activation, the current-voltage relationship, and the current inactivation (Fig. 3, C and D) were similar in PASMCs from wild-type and p62–/– mice (e.g., current density at +40 mV was 10.9 ± 1.3 and 11.6 ± 1.7 pA/pF, respectively; and at 200 ms, current decayed by 12.7 ± 2.8 and 14.3 ± 2.9%, respectively). As expected, U46619[GenBank] caused a significant inhibition in the whole range of channel activation and depolarized the membrane in PASMCs from wild-type mice (Fig. 3, A, C, and E). Current inactivation at +40 mV was also increased by U46619[GenBank] (i.e., at 200 ms, the current decayed by 21.7 ± 2.9%; p < 0.05). However, the TXA2 analog had no effect on KV currents in PASMCs from p62–/– mice (Fig. 3, B, D, and E).
Fig. 3. TP receptor activation leads to KV current inhibition and depolarization in PASMCs from wild-type (A, C, and E) but not from p62–/– (B, D, and F) mice. A and B, current traces for 200-ms depolarization pulses from –60 to +40 mV (in 10-mV increments) from a holding potential of –60 mV before (control) and after application of the TXA2 analog U46619 (100 nM). C and D, current-voltage relationship measured at the end of the 200-ms pulse (means ± S.E.M. of five to six cells). E and F, effects of U46619 on membrane potential recorded under current-clamp conditions.*, p < 0.05 versus control (paired Student's t test).
Basal levels of [Ca2+]i in p62–/– (184 ± 35 nM; n = 5) were not significantly different from those in wild-type mice (170 ± 45 nM; n = 6). We found that genetic inactivation of p62 abolished the increase in [Ca2+]i induced by U46619[GenBank] (Fig. 4, B and C). However, the contractile response induced by the two concentrations of U46619[GenBank] tested was remarkably similar in p62–/– and wild-type mice (Fig. 4D).
Fig. 4. PAs from p62–/– mice show no [Ca2+]i responses but preserved contractions induced by TP receptor activation. A and B, simultaneous recordings of [Ca2+]i (top trace) and force (bottom trace) in PA from p62+/+ and p62–/–, respectively, stimulated by 30 and 300 nM U46619. The averaged values (means ± S.E.M. of five PAs) of U46619-induced increase in [Ca2+]i and force are shown in C and D, respectively. *, p < 0.05 versus p62+/+ (unpaired Student's t test).
Role of KV1.5 Channels in TXA2-Induced Effects. KV1.5 channels have been reported to be major contributors of KV currents in PASMCs in several animal species. Figure 5A shows hKV1.5 current traces recorded in Ltk– cells stably expressing hKV1.5 channels. U46619[GenBank] (100 nM) significantly inhibited hKV1.5 currents. This inhibitory effect was only observed at the end of the depolarizing pulse; e.g., currents were almost unaffected at the peak (4.6 ± 2.4% decrease; not significant), but they were reduced by 17.8 ± 4.2% after 200 ms (n = 4; p < 0.05). In rat PASMCs, U46619[GenBank] also inhibited KV currents (Fig. 5B) as described previously (Cogolludo et al., 2003). The KV1.5 channel blocker DPO-1 (Lagrutta et al., 2006) inhibited KV currents in rat PASMCs. In the presence of DPO-1, U46619[GenBank] produced no further inhibitory effects (Fig. 5B). Therefore, we analyzed a possible interaction between PKC, KV1.5 channels and p62. Rat pulmonary arteries were incubated for 30 s in the absence (control) or presence of U46619.[GenBank] Homogenates were immunoprecipitated with anti-PKC or anti-KV1.5 antibodies, and the content of KV1.5, PKC, or p62 in the immunoprecipitates was analyzed via Western blot. Figure 5C shows that in immunoprecipitates of KV1.5 both PKC and p62 were present. The KV1.5-PKC and the KV1.5-p62 association were 135 ± 13% (n = 8; p = 0.06, not significant) and 163 ± 31% (n = 7; p < 0.05), respectively, in U46619[GenBank]-treated versus untreated arteries. The KV1.5-PKC interaction was also observed in immunoprecipitates of PKC immunoblotted with the anti-KV1.5 antibody (data not shown).
Fig. 5. Role of KV1.5 channels: TP receptor activation inhibits KV1.5 currents and increases coimmunoprecipitation of KV1.5 with PKC. A, current traces recorded in Ltk– cells stably transfected with human KV1.5 before (control) and after 100 nM U46619 and current-voltage relationship (means ± S.E.M. of four cells) measured at the end of the 200-ms pulse. Depolarizing steps from –60 to +60 mV were applied from a holding potential of –60 mV. *, p < 0.05 versus control (paired Student's t test). B, current traces recorded in rat PASMCs cells before (control) and after the 100 nM U46619 (top) or before (control), after DPO-1 (300 nM), and after DPO-1 plus U46619 (bottom). Current-voltage relationships are shown at the right (means ± S.E.M. of three to four cells). *, p < 0.05 versus control (paired Student's t test). C, rat pulmonary arteries were incubated for 30 s in the absence (control) or presence of 100 nM U46619, frozen, and homogenated. Homogenates were immunoprecipitated with anti-KV1.5 antibodies and immunoblotted with anti-PKC or anti-p62. Results are representative of samples from seven to eight mice. Each pair of bands (control and U46619) is obtained from the same animal.
Interaction of PKC with KV Channels: Role of p62. To determine the potential role of the PKC scaffold protein p62, the PKC-KV1.5 interaction was analyzed by coimmunoprecipitation in lungs from wild-type and p62–/– mice. Genetic inactivation of p62 in mice did not modify the expression levels of either PKC or KV1.5 channels in PASMCs (Fig. 6A). In immunoprecipitates of PKC from wild-type mice immunoblotted with the anti-KV1.5 antibody, a band of approx. 80 kDa was observed, which presumably reflects the mature (glycosylated) form of the channel expressed in the membrane (Li et al., 2000). However, p62-deficient mice showed a weak PKC-KV1.5 coimmunoprecipitation (Fig. 6B).
Fig. 6. Similar expression of PKC and KV1.5 but reduced interaction between PKC and KV 1.5 in p62–/– versus p62+/+. A, representative Western blots of lung homogenates using anti-KV1.5 and anti-PKC antibodies. B, lung homogenates were immunoprecipitated with anti-PKC antibodies and immunoblotted with anti-KV1.5; membranes were reblotted with the anti-PKC antibody as a loading control. The graph shows the densitometric analysis of the KV1.5 protein relative to PKC and expressed as a percentage of values in wild-type mice. **, p < 0.01 versus wild type.
By using nonselective PKC inhibitors and the PKC-selective inhibitor PKC-PI, we suggested that PKC was involved in the KV channel inhibition and the contractile response induced by TXA2 in rat pulmonary artery myocytes (Cogolludo et al., 2003, 2005). Herein, we confirmed the role of PKC in native KV currents by using PASMCs from PKC–/– mice. Consistent with the essential role of KV1.5 channels in the pulmonary vasculature, we show that the KV1.5 inhibitor DPO-1 inhibited KV currents in native rat PASMCs by approx. 50% and that the TXA2 analog U46619[GenBank] had no further inhibitory effects. In addition, cloned human KV1.5 channels expressed in Ltk– cells were also inhibited by U46619.[GenBank] Moreover, our results demonstrate the interaction between PKC and KV1.5 in both rat PAs and mouse lungs, which was minimal in p62–/– mice. Deletion of p62 abolished KV channel inhibition and Ca2+ responses induced by TXA2, further supporting the role of p62 as a key mediator between PKC and KV1.5. However, our study also showed that the contractile response induced by U46619[GenBank] in PA was similar in wild-type and p62–/– mice.
In both rat and newborn porcine PASMCs, U46619[GenBank] inhibited KV currents, depolarized cell membrane, increased [Ca2+]i through CaL channels, and induced a contractile response (Cogolludo et al., 2003, 2005). U46619[GenBank] had no direct effect on CaL channels in voltage-clamped cells, indicating that increased Ca2+ entry through CaL channels is secondary to membrane depolarization. Herein, we demonstrated that, in mice, U46619[GenBank] also inhibits KV currents in PASMCs and induces a [Ca2+]i response and vasoconstriction in isolated PA. The degree of KV channel inhibition in mice PASMCs (25% at 100 nM U46619[GenBank]) was similar to that observed in porcine and in rat PA, and it was accompanied by a significant membrane depolarization. In rat and porcine PAs, all these effects were inhibited by calphostin C and PKC-PI (Cogolludo et al., 2003, 2005). These experiments suggested a role for PKC as a link between TP receptors and KV channels, which was confirmed in the present study using PKC–/– mice. The magnitude and current-voltage relationship of KV currents were similar in the wild-type and knock-out animals, suggesting no changes in the channel proteins underlying KV currents. Thus, genetic inactivation or pharmacological inhibition of PKC abolished the effects of U46619[GenBank] on KV currents or membrane potential in PASMCs. In contrast, both approaches only partially inhibited (50–70%) the Ca2+ signal induced by U46619[GenBank] in rat and mice PAs, indicating that, in addition to the PKC-KV-CaL pathway, mechanisms increasing [Ca2+]i (e.g., Ca2+ release from intracellular stores) are also activated in response to U46619[GenBank] (Snetkov et al., 2006).
The present experiments also indicate that in mice, PKC contributes to the vasoconstriction induced by TP receptor activation. These results are in agreement with those obtained in rats and newborn piglets using PKC-PI (Cogolludo et al., 2003, 2005). However, in 2-week-old piglets (Cogolludo et al., 2005), PKC-PI and the Ca2+ channel blocker nifedipine almost fully inhibited U46619[GenBank]-induced increases in [Ca2+]i, but they had no effect on U46619[GenBank]-induced contractile responses; i.e., there was a contractile response in the absence of changes in [Ca2+]i. Therefore, in these animals, the up-regulation of Ca2+-independent mechanisms for contraction (Somlyo and Somlyo, 2000) makes PKC and the [Ca2+]i signal redundant.
KV currents recorded in native PASMCs reflect the contribution of multiple KV channel proteins [e.g., in human PAs, 22 transcripts of KV subunits: KV1.1 to KV1.7, KV1.10, KV2.1, KV3.1, KV3.3, KV3.4, KV4.1, KV4.2, KV5.1, KV6.1 to -6.3, KV9.1, KV9.3, KV10.1, and KV11.1, and three of KV subunits KV1 to -3 have been identified by reverse transcription-polymerase chain reaction]. However, KV1.5 subunits are thought to be major contributors of the native KV currents in PAs from different species, and their activity is regulated by vasoactive factors such as 5-hydroxytryptamine (Cogolludo et al., 2006) and hypoxia (Platoshyn et al., 2006). Therefore, we analyzed the effects of U46619[GenBank] on the KV current carried by human cloned KV1.5 channels expressed in mouse fibroblast (Ltk–) cells. This cell line expresses endogenously the KV2.1 subunit, which assembles with the transfected hKv1.5 protein (Uebele et al., 1996). U46619[GenBank] induced a weak but significant inhibitory effect on this current, suggesting that KV1.5 channels are involved in the effects of TP receptor activation in native PASMCs. The small inhibition in this cell type probably reflects a lower efficacy of the signaling pathway compared with rat or mouse PASMCs. Furthermore, after pharmacological inhibition of KV1.5 channels with DPO-1, U46619[GenBank] had no further inhibitory effects on KV currents in rat PASMCs.
In the present article, we show that PKC coimmunoprecipitates with KV1.5 channels. In a previous study (Cogolludo et al., 2003), we reported that U46619[GenBank] induced the translocation of PKC from the cytosolic to the membrane fraction. Therefore, TP receptor-induced KV channel inhibition is associated with the translocation of PKC to the plasma membrane where it interacts with KV1.5 channels. This PKC-KV1.5 interaction is not necessarily a direct protein-protein interaction; it seems more likely that it is mediated by adaptor proteins. In this regard, it has been described that PKC can interact with the subunit KV2 of the KV channel via the p62 adaptor protein (Gong et al., 1999). In immunoprecipitation experiments, we found that p62 was present in the KV1.5-PKC complex. Even when the complex was constitutive, the association of p62 with KV1.5 increased significantly by U46619.[GenBank] Furthermore, the PKC-KV1.5 coimmunoprecipitation was strongly reduced in p62–/– mouse lung, indicating that p62 physically associates PKC into the KV channel complex.
KV subunits function as molecular chaperones, and they can directly regulate channel inactivation, voltage dependence, and current amplitude (Martens et al., 1999). p62 overexpression stimulates PKC-dependent phosphorylation of KV2 (Gong et al., 1999), and it induces a hyperpolarizing shift of KV current activation in pheochromocytoma cells (Kim et al., 2004). Thus, we analyzed the effect of genetic inactivation of p62 on KV currents and its modulation by TP receptor activation. KV currents in PASMCs from p62–/– were similar to wild type. As expected, U46619[GenBank] had no effect on KV currents in p62–/– PASMCs, indicating that the p62-dependent PKC-KV1.5 interaction is required for the inhibitory effect of TP receptor activation on KV current.
Thus, genetic inactivation of p62 had a similar effect to genetic or pharmacological inactivation of PKC regarding KV current modulation. We were surprised to find that p62 gene deletion fully inhibited the Ca2+ response induced by U46619[GenBank] in isolated PAs compared with a 50 to 70% inhibition by PKC inactivation. More intriguingly, the contractile response to U46619[GenBank] was not affected in PA from p62–/– mice. This contractile response in the absence of changes in [Ca2+]i must then be attributed to Ca2+-independent mechanisms (i.e., Ca2+ sensitization; Somlyo and Somlyo, 2000). This response to U46619[GenBank] in p62–/– mice PA is similar to that observed in 2-week-old piglet PAs after inhibition of PKC (i.e., contraction without [Ca2+]i signal) (Cogolludo et al., 2005). In these animals, there is an up-regulation of Rho kinase (Bailly et al., 2004), a key enzyme in Ca2+-sensitizing mechanisms. In addition, Rho kinase inhibitors were more effective inhibiting U46619[GenBank] contractions in these piglets than in newborn piglets or adult rats (Cogolludo et al., 2005). Thus, we speculate that the chronic down-regulation of the PKC-p62-KV-CaL-dependent pathway, either at the level of KV channel activity (as occurs in older piglets) or p62 (p62–/– mice), but not PKC (PKC–/– mice), is compensated by up-regulation of Ca2+ sensitization mechanisms.
In conclusion, PKC modulates KV channel function, and it is involved in pulmonary vasoconstriction induced by TP receptor activation. The interaction between PKC and KV1.5 and the inhibitory effect of U46619[GenBank] in cloned human KV1.5 channels suggest that these specific channel subtypes are functional targets for PKC. The adaptor protein p62 is required for the PKC-KV1.5 interaction and hence for the inhibition of KV currents after TP receptor activation.
ABBREVIATIONS: KV, voltage-gated K+; PASMC, pulmonary artery smooth muscle cell; CaL, voltage-dependent L-type Ca2+ channel; TXA2, thromboxane A2; PH, pulmonary hypertension; TP, thromboxane-endoperoxide; PKC, protein kinase C; PA, pulmonary artery; IP, inhibitory peptide; aPKC, atypical PKC; PSS, physiological salt solution; h, human; U46619[GenBank], 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2; DPO-1, [1S-1,2,5]-[5-methyl-2-(1-methylethyl) cyclohexyl] diphenyl phosphine oxide.
【参考文献】
Archer S, Souil E, Dinh-Xuan AT, Schremmer B, Mercier JC, El Yaagoubi A, Nguyen-Huu L, Reeve HL, and Hampl V (1998) Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv1.2, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J Clin Invest 101: 2319–2330.[Medline]
Bailly K, Ridley AJ, Hall SM, and Haworth SG (2004) RhoA activation by hypoxia in pulmonary arterial smooth muscle cells is age and site specific. Circ Res 94: 1383–1391.[Abstract/Free Full Text]
Barnes PJ and Liu SF (1995) Regulation of pulmonary vascular tone. Pharmacol Rev 47: 87–131.[Medline]
Cogolludo A, Moreno L, Bosca L, Tamargo J, and Perez-Vizcaino F (2003) Thromboxane A2-induced inhibition of voltage-gated K+ channels and pulmonary vasoconstriction: role of protein kinase Czeta. Circ Res 93: 656–663.[Abstract/Free Full Text]
Cogolludo A, Moreno L, Lodi F, Frazziano G, Cobeno L, Tamargo J, and Perez-Vizcaino F (2006) Serotonin inhibits voltage-gated K+ currents in pulmonary artery smooth muscle cells: role of 5-HT2A receptors, caveolin-1, and KV1.5 channel internalization. Circ Res 98: 931–938.[Abstract/Free Full Text]
Cogolludo A, Moreno L, Lodi F, Tamargo J, and Perez-Vizcaino F (2005) Postnatal maturational shift from PKCzeta and voltage-gated K+ channels to RhoA/Rho kinase in pulmonary vasoconstriction. Cardiovasc Res 66: 84–93.[Abstract/Free Full Text]
Durán A, Serrano M, Leitges M, Flores JM, Picard S, Brown JP, Moscat J, and Diaz-Meco MT (2004) The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis. Dev Cell 6: 303–309.[CrossRef][Medline]
Frey RS, Gao X, Javaid K, Siddiqui SS, Rahman A, and Malik AB (2006) Phosphatidylinositol 3-kinase signaling through protein kinase C induces NADPH oxidase-mediated oxidant generation and NF-B activation in endothelial cells. J Biol Chem 281: 16128–16138.[Abstract/Free Full Text]
Gayral S, Deleris P, Laulagnier K, Laffargue M, Salles JP, Perret B, Record M, and Breton-Douillon M (2006) Selective activation of nuclear phospholipase D-1 by g protein-coupled receptor agonists in vascular smooth muscle cells. Circ Res 99: 132–139.[Abstract/Free Full Text]
Godeny MD and Sayeski PP (2006) G II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus. Am J Physiol Cell Physiol 291: C1297–C307.[Abstract/Free Full Text]
Gong J, Xu J, Bezanilla M, van Huizen R, Derin R, and Li M (1999) Differential stimulation of PKC phosphorylation of potassium channels by ZIP1 and ZIP2. Science 285: 1565–1569.[Abstract/Free Full Text]
Halushka PV, Mais DE, Mayeux PR, and Morinelli TA (1989) Thromboxane, prostaglandin and leukotriene receptors. Annu Rev Pharmacol Toxicol 29: 213–239.[CrossRef][Medline]
Hirai T and Chida K (2003) Protein kinase C (PKC): activation and cellular functions. J Biochem 133: 1–7.[Abstract/Free Full Text]
Kim Y, Park MK, Uhm DY, Shin J, and Chung S (2005) Modulation of delayed rectifier potassium channels by alpha1-adrenergic activation via protein kinase C zeta and p62 in PC12 cells. Neurosci Lett 387: 43–48.[Medline]
Kim Y, Uhm DY, Shin J, and Chung S (2004) Modulation of delayed rectifier potassium channel by protein kinase C zeta-containing signaling complex in pheochromocytoma cells. Neuroscience 125: 359–368.[CrossRef][Medline]
Krotova K, Hu H, Xia SL, Belayev L, Patel JM, Block ER, and Zharikov S (2006) Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation. Br J Pharmacol 148: 732–740.[CrossRef][Medline]
Lagrutta A, Wang J, Fermini B, and Salata JJ (2006) Novel, potent inhibitors of human Kv1.5 K+ channels and ultrarapidly activating delayed rectifier potassium current. J Pharmacol Exp Ther 317: 1054–1063.[Abstract/Free Full Text]
Leitges M, Sanz L, Martin P, Duran A, Braun U, Garcia JF, Camacho F, Diaz-Meco MT, Rennert PD, and Moscat J (2001) Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Mol Cell 8: 771–780.[CrossRef][Medline]
Li D, Takimoto K, and Levitan ES (2000) Surface expression of Kv1 channels is governed by a C-terminal motif. J Biol Chem 275: 11597–11602.[Abstract/Free Full Text]
Liu LZ, Zhao HL, Zuo J, Ho SK, Chan JC, Meng Y, Fang FD, and Tong PC (2006) Protein kinase Czeta mediates insulin-induced glucose transport through actin remodelling in L6 muscle cells. Mol Biol Cell 17: 2322–2330.[Abstract/Free Full Text]
Martens JR, Kwak Y-G, and Tamkun MM (1999) Modulation of Kv channel / subunit interactions. Trends Cardiovasc Med 9: 253–258.[CrossRef][Medline]
Moscat J and Diaz-Meco MT (2000) The atypical protein kinase Cs. Functional specificity mediated by specific protein adapters. EMBO Rep 1: 399–403.[CrossRef][Medline]
Pérez-Vizcaíno F, Cogolludo A, and Tamargo J (1999) Modulation of arterial Na+-K+-ATPase-induced [Ca2+]i reduction and relaxation by norepinephrine, ET-1, and PMA. Am J Physiol 276: H651–H657.[Medline]
Platoshyn O, Brevnova EE, Burg ED, Yu Y, Remillard CV, and Yuan JX (2006) Acute hypoxia selectively inhibits KCNA5 channels in pulmonary artery smooth muscle cells. Am J Physiol Cell Physiol 290: C907–C916.[Abstract/Free Full Text]
Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto K, Wang S, Moudgil R, Harry G, et al. (2003) In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation 107: 2037–2044.[Abstract/Free Full Text]
Remillard CV, Tigno DD, Platoshyn O, Burg ED, Brevnova EE, Conger D, Nicholson A, Rana BK, Channick RN, Rubin LJ, et al. (2007) Function of Kv1.5 channels and genetic variations in KCNA5 in patients with idiopathic pulmonary arterial hypertension. Am J Physiol Cell Physiol 292: C1837–C1853.[Abstract/Free Full Text]
Salamanca DA and Khalil RA (2005) Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension. Biochem Pharmacol 70: 1537–1547.[CrossRef][Medline]
Shimoda LA, Sylvester JT, Booth GM, Shimoda TH, Meeker S, Undem BJ, and Sham JS (2001) Inhibition of voltage-gated K+ currents by endothelin-1 in human pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 281: L1115–L1122.[Abstract/Free Full Text]
Shizukuda Y and Buttrick PM (2002) Protein kinase C-zeta modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt. Am J Physiol Heart Circ Physiol 282: H320–H327.[Abstract/Free Full Text]
Snetkov VA, Knock GA, Baxter L, Thomas GD, Ward JP, and Aaronson PI (2006) Mechanisms of the prostaglandin F2alpha-induced rise in [Ca2+]i in rat intrapulmonary arteries. J Physiol 571: 147–163.[Abstract/Free Full Text]
Somlyo AP and Somlyo AV (2000) Signal transduction by G-proteins, Rho kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185.[Abstract/Free Full Text]
Uebele VN, England SK, Chaudhary A, Tamkun MM, and Snyders DJ (1996) Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv2.1 subunits. J Biol Chem 271: 2406–2412.[Abstract/Free Full Text]
Valenzuela C, Delpon E, Tamkun MM, Tamargo J, and Snyders DJ (1995) Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers. Biophys J 69: 418–427.[Medline]
Ward JP, Knock GA, Snetkov VA, and Aaronson PI (2004) Protein kinases in vascular smooth muscle tone–role in the pulmonary vasculature and hypoxic pulmonary vasoconstriction. Pharmacol Ther 104: 207–231.[CrossRef][Medline]
Weir EK, Reeve HL, Huang JM, Michelakis E, Nelson DP, Hampl V, and Archer SL (1996) Anorexic agents aminorex, fenfluramine, and dexfenfluramine inhibit potassium current in rat pulmonary vascular smooth muscle and cause pulmonary vasoconstriction. Circulation 94: 2216–2220.[Abstract/Free Full Text]
Yuan XJ, Wang J, Juhaszova M, Gaine SP, and Rubin LJ (1998a) Attenuated K+ channel gene transcription in primary pulmonary hypertension. Lancet 351: 726–727.[CrossRef][Medline]
Yuan X-J, Wang J, Juhaszova M, Golovina VA, and Rubin LJ (1998b) Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle. Am J Physiol 274: L621–L635.[Medline]
作者单位：Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
1 From the Department of Epidemiology and Community Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada (YC); the Institute of Agricultural Rural and Environmental Health (DR and JD) and the College of Nursing (DR), University of Saskatchewan, Saskatoon, Canada; and the Centre de Pneumologie, Hôpital and Université Laval, Sainte-Foy, Canada (YFC)
2 Supported by grant no. 200203MOP-100752-POP-CCAA-11829 from the Canadian Institutes of Health Research. 3 Reprints not available. Address correspondence to Y Chen, Department of Epidemiology and Community Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada K1H 8M5. E-mail: ychen{at}uottawa.ca.
ABSTRACT
Background: Obesity is becoming a serious public health issue and is related to lung dysfunction. Because both weight and height are indicators of body size, body mass index (BMI) may not be an ideal index of obesity in prediction of pulmonary dysfunction.
Objective: The objective of the study was to determine the predictability of waist circumference (WC) and BMI for pulmonary function in adults with and without excess body weight.
Design: A cross-sectional study of 1674 adults aged 18 y was conducted in a rural community. Height, weight, WC, and pulmonary function were measured. Multivariate analysis was conducted.
Results: WC was negatively associated with forced vital capacity and forced expiratory volume in 1 s, and the associations were consistent across sex, age, and BMI categories. On average, a 1-cm increase in WC was associated with a 13-mL reduction in forced vital capacity and an 11-mL reduction in forced expiratory volume in 1 s. The association between WC and pulmonary function was consistent in subjects with normal weight, overweight, and obesity. In subjects with normal weight, BMI was positively associated with forced vital capacity and forced expiratory volume in 1 s.
Conclusion: WC, but not BMI, is negatively and consistently associated with pulmonary function in normal-weight, overweight, and obese subjects.
Key Words: Adults • body mass index • lung • lung function • obesity • waist circumference
INTRODUCTION
Obesity is becoming a serious public health issue, especially in developed countries (1). A growing body of evidence indicates that obesity is associated with a wide range of health conditions, including respiratory diseases such as chronic obstructive pulmonary disease (COPD; 2) and asthma (3). Numerous studies have examined the association between body mass index (BMI; in kg/m2) or weight change and pulmonary function testing variables, and the associations vary in different subpopulations (4-15).
Body weight and BMI can be easily measured and therefore are frequently used in large-scale epidemiologic studies. A major limitation of these measures is that they do not distinguish between fat mass and muscle (lean) mass, which have opposite effects on pulmonary function (7, 11, 16). Another important limitation is that both weight and height are surrogate measures of body size and are important predictors for pulmonary function measurements. A unit of body weight or BMI is likely to have less fat mass for underweight persons and for men than for overweight persons and for women (7). In addition, body weight and BMI provide no information on the nature of body fat distribution, both of which may play an important role on the association between obesity and pulmonary function (7).
Several studies have evaluated the relation of waist circumference (WC) and waist-to-hip ratio (WHR) to pulmonary function testing variables (14, 16-22). However, whether WC and BMI have a similar predictability for pulmonary function in nonobese and obese subjects is not known. This study aimed to determine the predictability of WC and BMI for pulmonary function in adults with and without excess body weight.
SUBJECTS AND METHODS
This analysis was based on data from a cross-sectional study conducted in the town of Humboldt, Saskatchewan, Canada, in 2003 (23). The target population was all residents of the town aged 18–79 y. Almost all of the study population was white. A total of 2057 adults—71% of the target population—participated in the study. The details of the study were given in a previous report (23).
Written informed consent was obtained from each participant. The study was approved by the University of Saskatchewan research ethics board.
Canvassers distributed a self-administered questionnaire to all eligible residents. Completed questionnaires were returned during a scheduled clinic visit. Collected information included demographic factors, education, occupation, income, smoking habits, coffee and alcohol consumption, respiratory symptoms, and illnesses. As defined previously (7, 23), current smokers were participants who reported smoking every day or almost every day and who had smoked 20 packs of cigarettes during their lifetime. Ex-smokers were those who had been regular smokers but who, at the time of the survey, had not smoked for 6 mo. Otherwise, subjects were defined as nonsmokers. Perceived level of physical activities was also recorded. Leisure-time physical activity was measured by asking the question: "Compared with the way other people your age now spend their spare time, would you say you are more physically active, equally physically active, or less physically active?"
During a clinical visit, lung function, height, weight, and WC were measured. Weight was measured to the nearest 0.1 kg. Height was measured (in cm) by using a fixed tape measure while participants stood, wearing no shoes, on a hard surface. WC was measured (in cm) horizontally through the narrowest part of the torso, between the lowest rib and the iliac crest (24).
A spirometer (MedGraphics CPF-S System; Medical Graphics Corporation, St Paul, MN) was used for pulmonary function testing. Two machines were calibrated every morning during the study period with the use of a standard syringe. Each subject was tested according to the American Thoracic Society criteria (25). Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and the ratio of FEV1 to FVC (FEV1:FVC, in %) were included in this analysis. Values were corrected for body temperature, pressure, and saturation with water vapor.
We examined the association between WC, BMI, and pulmonary function testing variables by using multivariate multiple regression analysis (WC and BMI as continuous variables) or multivariate analysis of variance (MANOVA) (WC and BMI as categorical variables). For WC, 3 pulmonary function indexes were considered simultaneously, and adjustments were made for sex, age, standing height, body weight, and pack-years of smoking (1 pack-year = 20 cigarettes · d–1 · y–1). Separate analyses were conducted for different BMI categories: normal weight, <25.0; overweight, 25.0–29.9; and obese, 30.0). Mean (95% CI) pulmonary function testing variables were calculated for subjects with low (<100 cm) and high (100 cm) WC after adjustment for height, weight, and pack-years of smoking. WC values >100 cm are most likely to be associated with potentially atherogenic metabolic disturbances in men and women (26). Similar analyses were performed for BMIs associated with the pulmonary function testing variables after adjustment for age and pack-years of smoking. Associations were considered to be significant if 2-sided P was < 0.05. All the analyses were conducted by using SPSS software (version 11.5; SPSS Inc, Chicago, IL).
RESULTS
The 1674 subjects (81.4% of the participants) who had satisfactory pulmonary function testing results and height, weight, and WC measures were included in this analysis. Of these 1674 subjects, 738 (45.1%) were men, and 936 (55.9%) were women. The mean (±SD) age was 51.0 ± 15.3 y for men and 50.4 ± 15.6 y for women. Of the study population, 35.0% were obese (BMI 30.0), a proportion that is higher than the Canadian national average (27), and 38.3% were overweight (BMI 25.0–29.9); 12.0% were current smokers, 33.8% were ex-smokers, and 54.2% were nonsmokers. The mean anthropometric measures and pulmonary function testing variables by sex and age groups are shown in Table 1.
View this table:
TABLE 1. Anthropometric measures and pulmonary function by sex and age1
WC was negatively associated with FVC and FEV1 after adjustment for both standing height and body weight (Table 2). On average, a 1-cm increase in WC was associated with a 13-mL reduction in FVC and an 11-mL reduction in FEV1 after adjustment for sex, age, height, weight, and pack-years of smoking. The association of WC with FEV1:FVC was not significant (P = 0.212). The adjusted means for FVC and FEV1 were significantly (P < 0.001) lower in subjects with a WC of 100 cm than in those with a WC of <100 cm (Figure 1).
View this table:
TABLE 2. Association between waist circumference and pulmonary function adjusted for covariates1
View larger version (11K):
FIGURE 1.. Adjusted mean (95% CI) pulmonary function testing variables by waist circumference in all subjects, obtained by multivariate ANOVA. FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s.
The association of WC and BMI with pulmonary function testing variables by BMI category is shown in Table 3. WC was negatively associated with FVC and FEV1, and the results were consistent across the age and BMI categories. The interaction of WC with BMI category was not significant for any of the pulmonary function testing variables. However, the association of BMI with pulmonary function was significantly modified by BMI category (Table 3). The associations of BMI with FVC and FEV1 were negative and significant in overweight and obese subjects. In subjects with normal weight (BMI <25.0), however, BMI was positively associated with FVC (P = 0.043) and FEV1 (P = 0.02). The pattern seen after further adjustment for height was similar.
View this table:
TABLE 3. Associations of BMI and waist circumference (WC) with pulmonary function testing variables by BMI category1
DISCUSSION
Our data show that WC is significantly associated with FVC and FEV1 but not with FEV1:FVC. On average, a 1-cm increase in WC was associated a 13-mL reduction in FVC (15 mL for men and 11 mL for women) and an 11-mL reduction in FEV1 (15 mL for men and 8 mL for women). The sex difference was not significant. These results are consistent with those from 2 previous epidemiologic studies. In a Scottish cross-sectional survey of 865 men and 971 women aged 25–64 y, Chen et al (18) found that WC was inversely associated with FVC (men: 8 mL/cm; women: 7 mL/cm) and FEV1 (men: 17 mL/cm; women: 9 mL/cm). In a British cohort study of 9674 men and 11 876 women aged 45–79 y, Canoy et al (17) found significant relations of WHR with FVC and FEV1 in both men and women. All of the above-mentioned associations persisted after adjustment for potential confounding factors. The current study also showed a tendency toward a stronger association between WC and FEV1, which is in line with previous observations (14, 20, 22).
An important observation of the current study is that WC consistently had a negative association with the pulmonary function testing variables in all BMI categories, whereas BMI was positively associated with FVC and FEV1 in normal-weight subjects. In a follow-up study of 3391 British subjects aged 18–73 y at baseline, Carey et al (14) found that the effect of weight gain on pulmonary function increased according to average weight at baseline.
For 2 major reasons, BMI is not an ideal measure for excess body weight as a predictor of pulmonary function. First, a higher BMI value for normal-weight persons than for obese persons may result from the fact that normal-weight persons have more muscle mass than fat mass. Second, BMI is calculated from body weight and height, which are correlated with body size—the larger the body size, the greater the pulmonary function testing variables. WC is also correlated with body size, but, when height and weight (which overall positively predicted pulmonary function testing variables) were included in the models, WC showed a consistently negative association with pulmonary function across the BMI categories. Our results indicated that WC as a measure of abdominal fat deposition has a somewhat more consistent predictability for pulmonary function. A recent study found that WC was a better predictor of pulmonary function than was BMI, although the study did not examine the associations in different BMI categories (28). Another study found that BMI was negatively, not positively, associated with mortality in elderly persons after adjustment for WC, whereas WC was positively associated with mortality after adjustment for BMI (29). The results of that study also suggest that, in an elderly population, WC is a better indicator of adiposity than is BMI. A recent study found that total body fat and central adiposity were inversely associated with lung function in elderly (30).
Several studies used WHR as a predictor of pulmonary dysfunction (14, 17-20). Canoy et al (17) compared the relations of WC, WHR, and BMI with FVC and FEV1 and found that pulmonary function was negatively associated with increasing quintiles of WHR, WC, and BMI, and, after adjustment for height, WHR in men and WC in women were associated with a bigger reduction in respiratory function than was BMI. Compared with WHR, WC is a more convenient measure, is less likely to be influenced by sex or degree of obesity (31), and is a better correlate with visceral adipose tissue (32). However, although these measures are convenient and can easily be applied in large-scale epidemiologic studies, they cannot separate out contributions of increased abdominal mass, restricted outward movement of the muscular abdominal wall, and intrathoracic factors. Magnetic resonance imaging (MRI) scanning, which can quantitate intraabdominal fat, may validate these measures in future epidemiologic studies.
Obesity is likely a cause of pulmonary function decline. Respiratory function is determined by the interaction of lungs, chest wall, and muscles. Truncal obesity reduces chest wall compliance, respiratory muscle function, and peripheral airway size (33-36). Findings of reductions in functional residual capacity, expiratory reserve volume, and vital capacity, particularly in patients with severe obesity, are consistent (37). Whereas smoking has a larger effect on expired flow rates (as reflected by FEV1) than on lung volume (as reflected by FVC), obesity affects lung volume to a larger degree than it affects expired flow rates. Our data showed that higher WC and BMI were associated with a significantly lower FVC, and consequently little effect on FEV1:FVC was seen, which suggests that obesity has a primary effect on lung volume. The mechanical effects of the intraabdominal pressure on the diaphragm are likely the main reason for the association of central obesity with compromised lung function. Abdominoplasty improves pulmonary function in healthy subjects (38).
Explanations other than a detrimental effect of obesity on respiratory function are less likely. When we adjusted for exercise (perceived level of activity compared with their peers), the results remained the same. Because smoking is related to lower body weight and worse pulmonary function, it is not likely to be an explanation for the association of excess weight and pulmonary function. The possibility exists that subjects in a sitting posture take slightly smaller inspirations—and therefore have lower FVC and FEV1 values—than do those in a standing posture (39). It is not known whether the comparative effects of sitting versus standing posture on the spirometric forced expiratory volumes are different in normal-weight, overweight, and obese subjects. However, even if those effects are different, those differences are unlikely to explain our results in the current study.
The clinical importance of the magnitude of association between WC and pulmonary function observed in the current study is not clear. However, numerous studies, including one from the same study population (23), have documented that abdominal obesity is associated with chronic respiratory disease.
In conclusion, our study found a consistent association between WC and pulmonary function in subjects with normal weight, overweight, and obesity. The negative association between BMI and pulmonary function was observed only in the overweight and obese. BMI was positively associated with FVC and FEV1 in normal-weight subjects. Intraabdominal pressure that has a mechanical effect on the diaphragm is suspected of being a major reason for the association of obesity with lung dysfunction.
ACKNOWLEDGMENTS
YC, DR, and JD contributed to the conception and design of the study; DR and JD supervised the data collection; YC performed the statistical analysis; YFC contributed to the explanation of the results; and all authors contributed to the writing of the manuscript. None of the authors had a personal or financial conflict of interest with any aspect of this research.
REFERENCES
1 From the Departments of Respiratory Medicine (MPKJE, EPAR, CLNDC, EFMW, and AMWJS) and Surgery (NEPD), Maastricht University, Maastricht, Netherlands
2 Supported by a grant from the European Dairy Association, Brussels, Belgium, and the Netherlands Asthma Foundation (NAF: 3.2.0034). 3 Reprints not available. Address correspondence to MPKJ Engelen, Department of Surgery, Maastricht University, PO Box 616, 6200 MD Maastricht, Netherlands. E-mail: mpkj.engelen{at}ah.unimaas.nl.
ABSTRACT
Background: Previously, we reported increased values for whole-body protein turnover in patients with chronic obstructive pulmonary disease (COPD) in the postabsorptive state.
Objective: The objective was to investigate whether intake of a carbohydrate-protein meal influences whole-body protein turnover differently in COPD patients and control subjects.
Design: Eight normal-weight patients with moderate COPD and 8 healthy control subjects were examined in the postabsorptive state and after 2 h of repeatedly ingesting a maltodextrin casein-based protein meal (0.02 g · kg body wt–1 · 20 min–1). Combined simultaneous, continuous, intravenous infusion of L-[ring-2H5]-phenylalanine and L-[ring-2H2]-tyrosine tracer and oral repeated ingestion of 1-13C-phenylalanine were performed to measure whole-body protein synthesis (WbPS) and first-pass splanchnic extraction of phenylalanine. Endogenous rate of appearance of phenylalanine as the measure of whole-body protein breakdown (WbPB) and netWbPS was calculated as WbPS – WbPB. Arterialized venous blood was sampled for amino acid enrichment and concentration analyses.
Results: Feeding induced an increase in WbPS and a reduction in WbPB. The reduction in WbPB was larger in the COPD group than in the control group (P < 0.05) and was related to the lower splanchnic extraction of phenylalanine in the patients. Consequently, netWbPS increased more after feeding in the COPD group than in the control group (P < 0.05).
Conclusion: Feeding induces more protein anabolism in normal-weight patients with moderate COPD than in healthy control subjects. This is probably because these COPD patients are characterized by an adaptive interorgan response to feeding to prevent or delay weight loss at this disease stage.
Key Words: Chronic obstructive pulmonary disease • protein feeding • first-pass splanchnic extraction • whole-body protein turnover • endogenous protein metabolism
INTRODUCTION
Muscle wasting commonly occurs in patients with chronic obstructive pulmonary disease (COPD), but different patterns of tissue depletion are observed. A substantial part of the COPD population is characterized by a normal weight with a shift in body composition toward reduced fat-free mass (FFM) despite a relative or absolute increase of fat mass (1, 2). In this group, functional capacity (ie, exercise capacity, muscle strength) and health status (3) are even more impaired than in the underweight patients with COPD with a relative preservation of FFM. This body-composition pattern is also seen with aging and could therefore be described as (accelerated) sarcopenia that could be reflected in altered whole-body substrate metabolism. Indeed, we showed a reduced ß-adrenoceptor–mediated lipolysis rate (4) and significantly higher amounts of whole-body protein turnover [protein synthesis (WbPS) and protein breakdown (WbPB) rates] in patients with COPD than in healthy, age-matched control subjects after overnight fasting (5). These data indicate that changes in intermediary metabolism are present in normal-weight patients with COPD that may trigger or reflect sarcopenia.
Although altered whole-body substrate turnover was observed in the postabsorptive state, no studies have yet examined the acute effect of feeding on substrate metabolism in COPD. Feeding is important because the fed state represents >50% of the 24-h metabolic activity and corresponds to the reconstitution of the protein lost during fasting. In COPD, the efficiency of maintaining body proteins may be declined as a result of a selective loss in the ability of skeletal muscle to efficiently use exogenous amino acids for protein anabolism. However, it is also possible that the splanchnic area is the compartment that is mainly contributing to the previously observed increased whole-body protein turnover in COPD (5, 6). The splanchnic tissues could limit the flow and the availability of alimentary amino acids to the peripheral tissues by influencing the absorption of the alimentary amino acids. In previous studies it has been shown that the first-pass splanchnic uptake of the amino acids leucine (7) and phenylalanine (8) increases with age. This means that if the splanchnic tissues use more amino acids, fewer amino acids will be available for the other (peripheral) tissues. Until now it was unknown whether chronic disease such as COPD further aggravated the age-related disturbances found in splanchnic extraction of amino acids, thereby negatively influencing the metabolic response to feeding in these patients.
Therefore, the purpose of the present study was to examine the response of whole-body protein turnover and splanchnic amino acid extraction to a given dose of a maltodextrin protein meal in patients with COPD. Milk-based protein (casein) was used because of its high nutritional value (protein quality) and because casein is the protein mostly used (and to the highest degree) in nutritional supplements.
SUBJECTS AND METHODS
Subjects
A group of 8 male patients with moderate airflow obstruction and 8 healthy male volunteers were studied. The patients had COPD according to American Thoracic Society guidelines (9) and chronic airflow limitation, defined as measured forced expiratory volume in 1 s (FEV1) < 70% of reference FEV1. Furthermore, the patients had irreversible obstructive airway disease (<10% improvement of FEV1 predicted baseline after inhalation of ß2-agonist) and were in clinically stable condition and had not experienced respiratory tract infection or exacerbation of their disease at least 4 wk before the study. The patients with COPD were outpatients, attending the hospital for routine pulmonary control every 6 or 12 mo. Exclusion criteria were malignancy, cardiac failure, recent surgery, and severe endocrine, hepatic, or renal disorder. Also, subjects who were using systemic corticosteroids within 3 mo before the beginning of the study were excluded. The number of present smokers in the COPD and control groups was 2. The number of former smokers in the COPD and control groups was 5 (average number of years stopped was 10.2) and 2 (average number of years stopped was 12.5), respectively. Body mass index (BMI; in kg/m2) was not significantly different between the groups (control group: 25.4 ± 0.9; COPD group: 27.2 ± 0.8). The maintenance treatment of the studied patients consisted of inhaled ß2 agonists, inhaled anticholinergics, inhaled corticosteroids, oral theophylline, or a combination. Written informed consent was obtained from all subjects, and the study was approved by the medical ethics committee of the University Hospital Maastricht.
Pulmonary function tests
All patients and healthy volunteers underwent spirometry to determine FEV1, and the highest value from at least 3 technically acceptable assessments was used. Diffusing capacity of the lung for carbon monoxide was measured by using the single-breath method (Masterlab; Jaeger, Wurzburg, Germany). All values obtained were related to a reference value and expressed as percentages of the predicted value (10).
Study protocol
The protocol started at 0715 after an overnight fast from at least 0000. All subjects were in the supine position for 3 h. After insertion of a catheter into the right antecubital vein, the first blood sample was taken for baseline measurements. Immediately thereafter, a primed-constant intravenous infusion of stable isotopes (80 mL/h) was started with the use of a calibrated pump (IVAC Corporation, San Diego, CA). Primed and constant infusion of the stable isotopes L-[ring-2H5]-phenylalanine(2H5-Phe; prime: 2.19 µmol/kg body wt; infusion: 0.053 µmol · kg FFM–1 · min–1) and L-[ring-2H2]-tyrosine (2H2-Tyr; prime: 0.95 µmol/kg body wt; infusion: 0.018 µmol · kg FFM–1 · h–1) were given through the catheter in the antecubital vein. Primed infusion of L-[ring-2H4]-Tyr (2H4-Tyr; 0.31 µmol/kg body wt) was given in addition through the same catheter. 1-13C-Phe was given orally in the postabsorptive state and together with the liquid meal every 20 min (prime: 0.88 µmol/kg body wt; infusion: 0.055 µmol · kg FFM–1 · min–1). Stable isotopes were purchased from Cambridge Isotopic Laboratories (Woburn, MA).
For sampling arterialized venous blood, a venous catheter was placed in a dorsal vein of the left hand, using the heated box technique (11), a technique to mimic direct arterial sampling. After 1.5 h of stable isotope infusion to reach steady state enrichments, enteral nutrition was started by sip feeding every 20 min, for a total duration of 2 h. The test meal involved a liquid casein-based protein meal and was given in an amount of 0.018 g · kg body wt–1 · 20 min–1. Total fluid intake was 0.67 mL · kg body wt–1 · 20 min–1 by enteral nutrition. Arterialized venous blood samples were taken at 80, 85, 90, 200, 205, and 210 min into infusion. Body composition was measured with the use of Bioelectrical Impedance Spectroscopy (BIS Xitron 4000B; Xitron Technologies, San Diego, CA) to express protein metabolism data per kilogram of FFM. FFM of the patients with COPD was calculated by using a patient's specific regression equation as described by Steiner et al (12), whereas FFM of the healthy control subjects was calculated by using a specific equation for elderly men as described by Lukaski et al (13).
Enteral protein meals
To avoid metabolic changes as a result of recent modifications of the diet, the subjects were instructed to eat their usual diet at least 3 d before the study. The dietary protein intake of the study subjects was ascertained retrospectively during 5 d by using the dietary history method (COPD group: 0.95 ± 0.10 g protein · kg body wt–1 · d–1; control group: 0.96 ± 0.07 g protein · kg body wt–1 · d–1).
The test meal on the experimental day contained 29.5 g sodium caseinate (casein protein meal: 4.0 g N) and 68.5 g maltodextrin dissolved in ultrapure water to 1000 mL fluid at 60 °C. In total, 301 mL enteral nutrition and 8.1 g protein (based on a 75-kg subject) was supplied during the study. The protein composition of the casein protein meal was a 1:1:1 mixture of commercially available French, Dutch, and Danish sodium caseinates. All meals were prepared at least 1 h before the start of the experiment. To ensure a complete dissolution of the proteins and to prevent bacterial growth, the meals were kept at 4 °C until use.
Sample processing
Analysis of arterialized venous blood
Promptly after sampling, blood was distributed in prechilled, heparinized tubes (Becton Dickinson Vacutainer System, Franklin Lakes, NJ) and kept on ice to minimize enzymatic reactions. All analyses were performed in plasma, obtained by centrifugation of whole blood at 4 °C for 10 min at 3120 x g. For amino acid analysis, 250 µL plasma was deproteinized by mixing it with 20 mg dry sulfosalicylic acid. For analysis of urea, glucose, lactate, and ammonia, 900 µL plasma was deproteinized by mixing with 90 µL of a 500 g/L trichloroacetic acid solution. All samples were stored at –80 °C until further analysis.
Biochemical analysis
The enrichments (tracer-to-tracee ratios) of the amino acids phenylalanine and tyrosine in arterialized venous plasma were analyzed by a liquid chromatography–mass spectrometry system (Thermoquest LCQ, Veenendaal, The Netherlands) (14). Plasma concentrations of amino acids were determined with the use of a fully automated HPLC (Pharmacia, Woerden, The Netherlands), after precolumn derivatization with o-phthaldialdehyde (15).
Plasma glucose, lactate, urea, and ammonia were analyzed spectrophotometrically on a COBAS Mira S (Roche Diagnostica, Hoffmann-La Roche, Basel, Switzerland) by standard enzymatic methods (16). Plasma insulin was analyzed with a commercially available electrochemiluminescence immunoassay (Hitachi Modular Analyzer; Roche, Mannheim, Germany).
Calculations
The sum of amino acids (SUM AA) represents the sum of measurable -amino acids (glutamine, glycine, threonine, histidine, citrulline, alanine, taurine, arginine, -amino butyric acid, tyrosine, valine, methionine, isoleucine, phenylalanine, tryptophan, leucine, ornithine, and lysine). All the metabolic data were determined under steady state conditions. Tracer:tracee of phenylalanine reached an isotopic steady state within 1.5 h of infusion and within 2 h of feeding (data not shown) in both groups.
In the postabsorptive and prandial state, WbPS is calculated as follows (5):
RESULTS
Eight male patients with COPD and 8 male healthy volunteers participated in the study (Table 1). Age, height, body weight, and BMI did not differ significantly between the groups, but a tendency toward a lower FFM index (NS) and higher fat mass index (NS) was found in the COPD group. In the control group, all lung function values were within the normal range. The patients with COPD were characterized by moderate airflow obstruction and a mildly reduced diffusing capacity for carbon dioxide. C-reactive protein concentrations tended (P = 0.095) to be higher in the COPD group than in the control group, probably because of the large range of C-reactive protein values (0.5–32 mg/L) in the COPD group.
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TABLE 1. . Characteristics of the study population1
Plasma concentrations of glucose, lactate, urea, and ammonia (Table 2) were not different in the postabsorptive state between the patients with COPD and the healthy control subjects. No group effect and no significant group-by-status interaction were observed for these variables. A status effect was observed for insulin and glucose (P < 0.001), indicating that feeding resulted in increased glucose and insulin concentrations. The concentration of urea and ammonia did not significantly change after feeding, although there was a tendency toward a reduction (P = 0.057 and P = 0.056, respectively).
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TABLE 2. . Plasma concentrations in arterialized blood in the postabsorptive state and during feeding1
No significant group-by-status interaction was observed for any of the variables for protein metabolism except for netWbPS (Table 3) . There was a significant group as well as a status effect for SPE and WbPS. SPE was significantly lower in the COPD group than in the control group (P < 0.001), whereas WbPS was higher in the COPD group (P < 0.05). Moreover, feeding resulted in a decrease in SPE (P < 0.05) and an increase in WbPS (P < 0.05). The feeding-induced increase in WbPS (WbPS) was not different between the COPD and the control groups. WbPB (Raend-Phe; P < 0.01) was lower after feeding. The feeding-induced reduction in WbPB (WbPB) was significantly larger (P < 0.05) in the COPD group than in the control group. As a consequence, feeding resulted in a significant increase in netWbPS (P < 0.001). The increase in netWbPS (netWbPS) was higher in the COPD group than in the control group (P < 0.05), resulting in higher absolute values for netWbPS in the prandial state in the COPD group than in the control group (P < 0.05).
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TABLE 3. . Measures of protein metabolism in the postabsorptive state and during feeding1
Phenylalanine concentration and phenylalanine clearance were not different between the COPD and control groups in the postabsorptive state. No significant group-by-status interaction was observed for both variables. There was a status effect for phenylalanine concentration (P < 0.001), indicating that feeding resulted in an increase in phenylalanine concentration. The increase in phenylalanine concentration after feeding (Phe conc) was higher in the control group than in the COPD group (P < 0.05). These findings were also present for SUM AA (data not shown). There was a status effect (P < 0.001), and, in addition, there was a tendency toward a difference in SUM AA between the COPD and control groups (P = 0.085). A group effect was observed for phenylalanine clearance (P < 0.01), indicating that phenylalanine clearance was lower in the COPD group than in the control group.
DISCUSSION
The ability to obtain homeostatic regulation of protein metabolic processes during the day is important to preserve muscle mass and to function long term. Insight into the protein metabolic response to feeding is of importance in COPD because low-intensity exercise has been shown to induce an increased amino acid release from muscle (19). This finding suggests that physical activity in daily life may induce protein catabolism in COPD. To maintain protein balance on a daily basis and to prevent muscle wasting in COPD for the longer term, a positive protein metabolic response to feeding is therefore of crucial importance. In the present study, feeding increased net WbPS to a higher extent in normal-weight patients with moderate COPD than in healthy control subjects, indicating an enhanced anabolic response to feeding in this patient group.
Effect of feeding on WbPS
Feeding induced an increase in WbPS, which is in line with data obtained in previous studies that showed a positive effect of mixed feeding on protein synthesis (20, 21). In the present study, 0.11 g protein/kg body wt was ingested in 2 h. On the basis of the fed state of 16 h/d, 0.87 g protein/kg body wt will be ingested, which is in line with the current recommended dietary allowances in the elderly (0.8 g protein · kg body wt–1 · d–1) (22) and slightly lower than the recorded daily dietary protein intake of the study groups. Earlier, it has been shown that to increase peripheral protein synthesis, high amino acid availability is important (23, 24). We observed a status effect for protein synthesis and the concentration of phenylalanine and SUM AA, indicating that feeding increased systemic amino acid availability and protein synthesis. However, despite the lower feeding-induced increase in the phenylalanine concentration in COPD, the increase in protein synthesis was not different between the groups.
First-pass splanchnic extraction of phenylalanine
The splanchnic tissues play an important role in the regulation of protein turnover because these tissues are responsible for absorption of the alimentary amino acids and their release to the peripheral tissues. In a study that compared elderly subjects with young healthy subjects, first-pass SPE of dietary leucine was twice as high in the elderly as in the young men (7). In line, a study by Volpi et al (8) showed that the SPE of oral phenylalanine was higher in the elderly than in the young. The exact reason for the elevated SPE of amino acids in the elderly is still unknown. However, it is believed that it may contribute to the development of sarcopenia because it reduces amino acid availability to the periphery.
We also measured SPE of phenylalanine after 2 h of feeding in the patients with COPD and the healthy control subjects using free 1-13C-Phe given orally and together with the liquid meal. Because the meal as well as the oral tracers was administered in the same continuous feeding protocol, no differences in absorption kinetics between phenylalanine in the meal and the oral 1-13C-Phe are expected. The data of Volpi et al (8) on SPE of phenylalanine in the healthy elderly are a bit higher than ours (47 ± 3% compared with 35 ± 7%). However, the meal composition used in the 2 studies was different (oral amino acid mixture compared with maltodextrin protein meal). Interestingly, there was a group effect for SPE of phenylalanine. SPE was lower in the patients with COPD than in the control group, indicating that there is lower phenylalanine extraction by the gut, liver, or both during feeding in the patients, which could lead to a higher peripheral availability of dietary phenylalanine. Therefore, it was expected that the lower SPE in COPD would induce a higher prandial phenylalanine concentration in these patients. In the present study, a feeding effect for phenylalanine concentration but no group effect was observed. Remarkably, the increase in phenylalanine concentration after feeding was lower in the COPD group than in the control group. As systemic phenylalanine concentration is mainly the result of the capacity of phenylalanine utilization for protein synthesis and hydroxylation, this finding suggests that, besides an increased phenylalanine release in the circulation, there is an increased phenylalanine removal from the circulation in COPD. In contrast, phenylalanine clearance was lower in the COPD group than in the control group and was not affected by feeding. We do not have a good explanation for this observation.
The lower SPE of phenylalanine in the COPD group was associated with a larger reduction of endogenous Ra of phenylalanine after feeding. Endogenous Ra of phenylalanine allows an accurate estimation of WbPB, because dietary phenylalanine sequestered by splanchnic tissues during the first pass cannot reach the metabolic pool where 2H5-Phe is infused. The data suggest that the lower SPE in COPD positively influences their anabolic response to a given meal, and that the metabolic efficiency of feeding is therefore larger in the COPD group than in the control subjects.
Possible factors inducing a lower first-pass splanchnic extraction in COPD
At present, we can only speculate about possible mechanisms of the reduced SPE in COPD. Besides an adaptation to increased needs in the body elsewhere as mentioned previously, it is also possible that the reduced SPE in COPD is reflecting a reduced splanchnic protein turnover rate rather than a reduced splanchnic amino acid net utilization. However, the possibility that the splanchnic (liver + gut) protein turnover is reduced in COPD is remarkable when considering that this patient group is generally characterized by a low-grade systemic inflammatory state (25). In line, C-reactive protein concentrations tended to be higher in the studied patients with COPD than in the control subjects. However, because inflammation is associated with an increased hepatic protein synthesis (26), one should expect an elevated (but not reduced) protein synthesis in the splanchnic liver compartment in COPD. Other factors known to influence splanchnic protein turnover are nicotine use and intake of certain drugs. Nicotine can act as a splanchnic circulation constrictor because it has been shown that smoking aggravates liver injury and that intraportal nicotine infusion in rats decreases hepatic blood flow (27). However, smoking status and history were not different between the COPD and control groups. The studied patients were clinically stable for at least 3 mo before the study, exhibiting normal blood gases and only using inhalation medication. Still, it is important to highlight that this patient group is regularly experiencing an acute exacerbation of the disease, which is characterized by an increased inflammatory state, changes in blood gases, and use of systemic medication (ie, oral corticosteroids and antibiotics). Nonsteroidal anti-inflammatory drugs are known to reduce blood flow in the splanchnic region (28). Acute changes in the arterial partial pressures of oxygen and carbon dioxide do not reduce splanchnic blood flow (29, 30) but together with an increased inflammatory state may induce changes in insulin sensitivity and thus influence protein metabolism. A positive association has been found between SPE of dietary leucine and BMI (7). Currently, no relation was found between SPE of dietary phenylalanine and body weight or composition. However, it is important to notice that only normal-weight patients with COPD were studied without evidence of muscle wasting.
More research is warranted to get insight into the underlying factors responsible for the lower SPE of amino acids in COPD. The gut plays an important role as buffer of amino acids during fasting (31). The elevated initial release of amino acids into the circulation in COPD may lead to a reduced buffer of amino acids in a later (fasting) phase. Measurement of protein kinetics after 2 h of feeding is therefore necessary in COPD to examine whether protein balance after this anabolic phase is still positive.
In conclusion, the anabolic response to feeding is higher in weight-stable moderate patients with COPD than in healthy control subjects. This is related to lower first-pass SPE in COPD, resulting in a larger reduction of WbPB after feeding. This study shows that normal-weight patients with COPD are characterized by a pronounced adaptive interorgan response to feeding, apparently sufficient to prevent or delay weight loss in this stage of their disease. More studies are needed to investigate whether this adaptive response to feeding is inadequate or absent in weight-losing patients with COPD.
ACKNOWLEDGMENTS
MPKJE was responsible for the study design, data collection, data analysis, and writing of the manuscript; EPAR and CLNDC were responsible for the data collection; EFMW and AMWJS were responsible for the study design and for reviewing the manuscript; NEPD was responsible for the study design and data analysis and for reviewing the manuscript. None of the authors had a financial or personal interest in any company or organization sponsoring the research, including advisory board affiliations.
REFERENCES
1 From the Departments of Infectious Diseases (AS, GEG, and DCM) and Chest Medicine (CFJR), St George’s Hospital Medical School, and the Department of Clinical Dietetics, St George’s Healthcare NHS Trust (LH), London.
2 Supported by the Wellcome Trust (International Fellowship to AS) Medical Research Council and Serono International SA (DCM). 3 Address reprint requests to A Schwenk, Department of Infectious Diseases, Royal Free Hospital, Hampstead, London NW3 2QG, United Kingdom. E-mail: a.schwenk{at}doctors.org.uk.
ABSTRACT
Background: Pulmonary tuberculosis is the classic cause of "consumption," but the pathogenesis of such wasting is largely unknown. Animal studies in other conditions suggest that leptin may be a mediator between proinflammatory cytokine activity and wasting.
Objective: We tested whether the leptin concentration, after control for body fat mass, is higher during active pulmonary tuberculosis than after recovery and whether it correlates with energy metabolism and proinflammatory cytokine activity.
Design: Nondiabetic adults with pulmonary tuberculosis (n = 32) were recruited into a prospective observational study. Patients found to be antibody positive for human immunodeficiency virus were excluded from the study. Dual-energy X-ray absorptiometry, indirect calorimetry, and food intake protocols were performed at baseline and after 1 and 6 mo of tuberculosis treatment. Fasting plasma leptin, tumor necrosis factor and its soluble receptor, and interleukin 6 were measured by enzyme-linked immunosorbent assay.
Results: Resting energy expenditure was close to Harris-Benedict predictions and did not change significantly during treatment, but energy intake increased. Leptin concentration was correlated in a log-linear fashion with percentage body fat but was independent of cytokines and energy intake. There was no significant difference in leptin, corrected for energy balance and fat mass, at baseline and after 1 and 6 mo of treatment.
Conclusions: These data are compatible with recovery from anorexia or starvation without discernible hyper- or hypometabolism. The close correlation of leptin with body fat mass is similar to observations in healthy subjects. No additional influence of disease state or proinflammatory cytokine activity was found. Leptin does not appear to be a component of the immune response to human pulmonary tuberculosis, and thus it cannot account for the weight loss and anorexia associated with tuberculosis.
Key Words: Basal metabolism • body composition • cytokines • densitometry • X-ray • energy intake • interleukin 6 • leptin • receptors • tumor necrosis factor • tuberculosis • wasting syndrome
INTRODUCTION
Several million persons will experience wasting this year because they are among the 8–12 million persons per year worldwide who are newly diagnosed with tuberculosis (1). In an unselected US cohort of patients diagnosed with tuberculosis, 45% lost weight and 26% had anorexia (2). In resource-poor countries, weight loss is an almost invariable sign of tuberculosis (3). Co-infection with human immunodeficiency virus (HIV; 4) and delayed diagnosis of tuberculosis contribute to the burden of wasting in resource-poor settings. Although antimycobacterial treatment often induces weight gain, patients may remain underweight even 6 mo after the initiation of successful chemotherapy (5). Despite the scale of the problem, the pathogenesis of wasting in tuberculosis is largely unknown (6).
Proinflammatory cytokines are prime candidates as causative agents of the metabolic changes that eventually result in tuberculosis-associated wasting (7). However, evidence for such a link between the immune response and wasting is equivocal and incomplete, as recently reviewed (6). In the search for other mediators, leptin has emerged as a new candidate.
Leptin is best known as a key mediator of energy metabolism, and it reports the status of body energy stores to feeding centers in the hypothalamus (8). In addition, leptin is now recognized as both a recipient and an effector of immune stimuli, belonging to the same class of cytokines as interleukin 6 (IL–6; 9, 10). It has been suggested that leptin mediates anorexia in chronic inflammatory states (11). In patients with pulmonary tuberculosis, a recent study found increased leptin concentrations and a correlation with increased concentrations of tumor necrosis factor (TNF-; 12).
In this report, we present leptin, body composition, and energy metabolism data from a prospective observational study of patients with pulmonary tuberculosis. The primary hypothesis was that, after control for fat mass, leptin would be higher in patients during active tuberculosis than after their recovery. Furthermore, we hypothesized that higher leptin concentrations would correlate with increased proinflammatory cytokine activity, increased resting energy expenditure (REE), and reduced food intake.
SUBJECTS AND METHODS
Study design and subjects
In this prospective longitudinal study, measures of energy metabolism, body composition, and immune response were assessed at 3 time points: within 3 d of the initiation of antimycobacterial treatment, after 1 mo of treatment, and after 6 mo of treatment. Patients (n = 32) with a clinical diagnosis of pulmonary tuberculosis were recruited from 2 London hospitals. Diagnosis was based on standard microbiologic or clinical criteria, ie, acid-fast bacilli in sputum microscopy, Mycobacterium tuberculosis in sputum culture, or a combination of typical signs on chest X-ray, typical clinical symptoms, and clinical response to empirical antimycobacterial treatment. Patients were required to be 18 y old and able to give written informed consent.
Patients were excluded if HIV co-infection was documented but were allowed to enter the study if the HIV status was still unknown. Patients who had undergone surgery, pregnancy, or childbirth < 2 mo before the study were excluded. Further exclusion criteria were severe renal, hepatic, or cardiac insufficiency; diabetes mellitus; or intake of corticosteroids at baseline. Patients with a final primary diagnosis other than tuberculosis and those later diagnosed with HIV co-infection were excluded from the data analysis. Healthy subjects recruited from staff and students of St George’s Hospital Medical School [7 women, 4 men; age: 23 ± 5 y; body mass index (in kg/m2): 22.5 ± 3.0] served as a control group for REE only, as described below and elsewhere (13).
Measurement methods
A heparinized blood sample was taken around 0900 after an overnight fast and centrifuged at 1215 x g for 30 min at 4 °C and then at 2380 x g for 15 min at 4 °C to obtain platelet-poor plasma, which was immediately frozen at -80 °C. Repeated freeze-thaw cycles were avoided. Leptin, IL-6, TNF-, and soluble TNF- receptor type 1 (sTNFR-1) were measured by enzyme-linked immunosorbent assay with the use of paired antibodies (R&D Systems, Abingdon, United Kingdom). All measurements were done in triplicate. REE was measured by indirect calorimetry on a Deltatrac calorimeter (Datex-Ohmeda, Helsinki). Gas calibration was performed before each measurement. To prevent cross-infection in patients with active pulmonary tuberculosis, the tube connecting the canopy to the calorimeter was replaced with a single-use disposable polyethylene tube attached to an airway filter (Ultipor BB50TE; Pall Corp, Newquay, United Kingdom). A pilot study in the 11 healthy controls found that, although flow was reduced by 40%, these changes in the Deltatrac setup did not introduce a systematic error. Validation of this method is described in a separate report (13). REE was compared between patients and controls and with values predicted by the Harris-Benedict equation (14). Food energy intake was assessed by 24-h recall protocols and a food-frequency questionnaire covering the previous wk. The higher estimate of energy intake from these 2 methods was used for further analysis. Professional interpreters were available during the assessment for any subjects who were not fluent in English.
Body weight was measured on a calibrated scale to the nearest 0.1 kg while the patient was wearing light clothing. The weight of such clothing, calculated from the average of 10 measurements of each standard clothing item, was subtracted. Height was measured on a calibrated wall-mounted scale to the nearest 0.1 cm. Body fat was determined by dual-energy X-ray absorptiometry (Lunar DPX; Aura Scientific, Milton Keynes, United Kingdom). The local research ethics committee approved the study, and the rules of the Helsinki Declaration (revision of 1983) were followed.
Statistical analysis
Because of skewed distribution, concentrations of leptin, IL-6, and TNF- were log transformed for further statistical analysis. Prior weight loss was categorized as 10%, < 10%, and unknown. The ratio between reported energy intake and measured REE was used as a variable of energy balance. Changes between time points (ie, baseline, 1 mo, and 6 mo) were tested by repeated-measures analysis of variance with sex, time, and sex-by-time interaction as independent variables. Data are given as mean (± SD) unless indicated otherwise. The correlation between fat mass and leptin was tested by analysis of variance, with controls for time and sex. Two methods were used to test whether associations between leptin and values of proinflammatory cytokine response or energy metabolism were partly independent from fat mass and energy balance. For continuous variables, this testing was done by partial correlation, calculated separately for each time point. For categorial variables (time points, sex, prior weight loss), this testing was done by calculating the residuals for leptin, ie, the difference between measured and predicted log10 (leptin), from linear regression against percentage body fat and energy balance. Residuals were then compared between time points or between subgroups at each time point by paired or unpaired t test. Significance testing was adjusted for multiple comparisons with the use of the Bonferroni method. Statistics were calculated with SPSS software, version 10.0 (SPSS Inc, Chicago; Internet: http://www.spss.com).
RESULTS
Baseline characteristics
Between June 1999 and November 2000, 102 patients with a clinical diagnosis of pulmonary tuberculosis were screened for eligibility. Diagnosis was confirmed in 80 of them, but 24 had concomitant diseases that excluded them from the study (5 diabetes, 11 HIV co-infection, and 8 other). Sixteen patients did not give informed consent. Therefore, 40 patients entered the study, 32 (80%) of whom completed all assessments. One patient died of a cause unrelated to tuberculosis, and 7 patients withdrew for personal reasons. The final analysis was therefore based on 32 patients, 23 (72%) of whom were male. The age of the subjects was 42.5 ± 21.1 y (range: 18–84 y). The racial or ethnic background was South Asian, white, African, and Hispanic in 18, 9, 4, and 1 patients, respectively. All patients but one were considered to have made a satisfactory clinical response to anti-tuberculous chemotherapy at the end of 6 mo, and none were found to have multi-drug–resistant mycobacteria. One patient developed new pulmonary infiltrates in the 4th mo of antimycobacterial treatment but recovered during an extended 9-mo course of treatment.
Nutritional status
The body mass index at baseline was 19.7 ± 2.6 (range: 15.2–24.5). A weight history could be obtained in 28 patients, 26 (92.9%) of whom reported a weight loss during the previous year of 11.6 ± 6.7% of their usual body weight (range: 1.3–26.5%). Weight loss had first been noted 151 ± 70 d before baseline (range: 16–724 d).
Clinical recovery was accompanied by increases in weight and body fat, both in absolute values and in percentage of body weight (Table 1). These changes tended to be more pronounced in men than in women, although this sex difference was not statistically significant (Table 1). Longitudinal gain over 6 mo amounted to 9.4 ± 8.4% for weight (ranging from 4.7% loss to 33.3% gain) and 41.7 ± 3.1% for body fat (ranging from 27% loss to 238% gain). This weight gain represented recovery of most of the weight previously lost: patients reached 93.1 ± 6.9% of their pre-illness weight after 1 mo and 99.0 ± 8.8% after 6 mo.
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TABLE 1 . Body composition and leptin concentrations during treatment of tuberculosis, by group1
Energy metabolism
As expected, absolute REE was higher in men than in women (5.8 ± 0.7 and 4.9 ± 1.0, respectively, at baseline; P < 0.001). However, neither changes in REE during treatment nor REE results normalized for age, sex, height, and weight (Harris-Benedict prediction; 14) or for fat-free mass differed significantly between men and women. Therefore, all further data are presented for the sexes combined (Table 2). REE did not differ significantly from the value predicted by Harris-Benedict at baseline and at 1 mo, but it was significantly (P < 0.01) lower than predicted at 6 mo (Table 2). Because measured REE was also significantly lower than predicted in 11 healthy controls (92.9 ± 9.1%, P < 0.05), REE as a percentage of the predicted value did not differ significantly between healthy controls and patients at any time point. REE was highly reproducible for individual patients, differing +3.8 ± 2.8% from the individual baseline at 1 mo and -1.7 ± 0.3% at 6 mo. Likewise, REE did not change between time points when expressed per kilogram of fat-free mass (Table 2).
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TABLE 2 . Cytokines and energy metabolism during treatment of tuberculosis1
By contrast, energy intake increased significantly during treatment (Table 2). Intra-individual increases were 35 ± 8% from baseline to 1 mo and 44 ± 17% from baseline to 6 mo (P < 0.01).
Cytokines
Plasma IL-6, sTNFR1, and C-reactive protein were above the reference range at baseline, and they decreased by 1 mo and 6 mo, without a difference between the sexes (Table 2). By contrast, no change in plasma TNF- concentration was found.
Leptin
Fasting plasma leptin concentrations increased between baseline and 1 mo and 6 mo (Table 1). The median increase in leptin in individual patients amounted to 42.9% during the first month and to 84.7% over the entire 6-mo treatment. Leptin concentration and percentage fat mass (%FM) were strongly correlated, following a log-linear curve (Figure 1). With each increase in %FM, leptin was estimated to increase by a factor of 1.043 (95% CI: 1.037, 1.051), after we controlled for time and sex. This corresponds to a doubling in leptin concentration for each 16% increment in %FM. Women had higher leptin concentrations than did men (Table 1).
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FIGURE 1. . Correlation between leptin concentration and percentage fat mass. Scatterplots of leptin concentration (logarithmic scale) against percentage fat mass at 3 time points (n = 32 each) are combined in this graph: baseline (), 1 mo (), and 6 mo (). Correlation coefficients were r2 = 0.55, r2 = 0.80, and r2 = 0.84 at baseline, 1 mo, and 6 mo, respectively. Regression lines did not differ significantly between time points.
Patients with weight loss > 10% were compared with those with lesser degrees of weight loss. Patients with such a degree of wasting had lower baseline leptin concentrations than did patients with lesser degrees of weight loss (6.4 ± 7.3 and 9.0 ± 9.9 x 10-6 g/L, respectively; P = 0.02); they also had significantly lower baseline %FM (P = 0.04) but similar concentrations of circulating cytokines and similar values for energy metabolism (data not shown).
Correlations between leptin, cytokine concentrations, and energy values were explored with and without control for %FM and energy balance and at baseline and 6 mo (Tables 3 and 4). No further significant correlations between these variables were found at 1 mo (data not shown). Log(leptin) was not significantly correlated to any of the cytokines. Neither REE nor reported food intake was correlated with leptin in this study. After control for %FM and energy balance by calculation of the residual leptin concentration, no significant difference between time points was found. Residual leptin at 1 mo was 1.27 times (95% CI: 0.94, 1.71) the baseline value, and at 6 mo, it was 1.23 times (95% CI: 0.91, 1.68) the baseline value. Residual leptin concentration also did not differ at any time point between men and women or between patients who did and patients who did not have weight loss > 10%. Changes in log(leptin) and changes in %FM did not correlate between baseline and 1 mo, but they were found to be strongly correlated between 1 mo and 6 mo (r 2 = 0.74, P < 0.001). Thus, %FM and energy balance were the only variables found to be associated with leptin, whereas disease status and cytokine response to tuberculosis were independent from leptin.
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TABLE 3 . Pearson’s bivariate (top right triangular area) and partial (bottom left triangular area) correlation coefficients (r) between leptin, proinflammatory cytokines, and energy balance at baseline1
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TABLE 4 . Pearson’s bivariate (top right triangular area) and partial (bottom left triangular area) correlation coefficients (r) between leptin, proinflammatory cytokines, and energy balance at 6 mo1
DISCUSSION
This study provides no support for the concept that leptin is the missing link between immune defense and wasting in pulmonary tuberculosis. Leptin concentrations closely reflected %FM and were not correlated with the proinflammatory cytokine response in active tuberculosis. No additional influence of disease activity or inflammatory cytokines was found.
In theory, leptin would have been an intriguing candidate for this role. Leptin concentrations closely reflect %FM and energy balance (15), but a growing number of additional roles in neuroendocrine regulation are being recognized for this hormone (16, 17). Leptin is both a recipient of and a stimulus for immunologic signals. In animal models, its production increases in response to bacterial lipopolysaccharide (10, 11) and turpentine (9). Its molecular structure is similar to that of the IL-6 cytokine family (18). Furthermore, it has a trophic effect on components of the immune system. The immunosuppression induced by starvation in mice is abolished if leptin is substituted (19), including stimulation of a Th1 pattern of proinflammatory cytokine production (20).
Evidence for such a role for leptin in humans is equivocal. Ex vivo stimulation of human peripheral blood mononuclear cells with leptin results in a Th1 pattern of cytokine release (21). Increased leptin concentrations were found in patients with sepsis (22–24). It is not known to what extent such findings are explained by hyperglycemia and hyperinsulinemia, which are common findings in such patients and a major stimulus for leptin production (25). Leptin concentration increases during acute surgical stress but returns to normal within 24 h (26–29). In patients with the AIDS wasting syndrome, leptin concentration was reduced, but this finding was explained by lower %FM rather than by the presence of HIV infection per se (30, 31). Similar findings were reported in patients with cancer-related cachexia (32). Finally, people with genetic leptin deficiency have largely normal immune function (33). Taken together, these data suggest that the leptin system reacts to acute metabolic stress but does not to contribute to chronic wasting in humans.
Leptin has been suggested as a component of a Th1 pattern of cytokines, the same pattern that is required for protective immunity against M. tuberculosis (34). The production of interferon and TNF- is crucial to the host defense against tuberculosis (35–37), but it may also be associated with anorexia and fever (38). Stimulation of leptin production during active tuberculosis may therefore contribute to wasting, in concert with other proinflammatory cytokines. Indeed, one study found higher leptin concentrations in patients with active pulmonary tuberculosis than in healthy controls, and leptin increased further during treatment (12). A positive correlation between TNF- and leptin concentrations was found at baseline. However, both effects were significant only in 8 female patients, and not in 22 male patients. Body fat mass was estimated from BMI rather than being directly measured in that study (12). Such estimates are of very limited validity (39).
By using dual-energy X-ray absorptiometry as an accurate measurement of body fat, we found a close correlation between leptin and %FM, which followed a curve similar to that found in healthy subjects (40). Correlations between leptin concentration and values of energy metabolism gave inconsistent results. The weak negative correlation between leptin concentration and REE and the lack of an association with energy intake are at odds with findings of other studies in which leptin was strongly influenced by recent energy balance (41).
Because of the short-lived and paracrine effects of TNF- itself, concentrations of sTNFR-1 were used as a marker of TNF- activity (38, 42). The sTNFR-1 concentrations decreased with recovery from tuberculosis, as previously shown in patients co-infected with M. tuberculosis and HIV (43). Concentrations of sTNFR-1 and leptin were not correlated to each other in the present study. By contrast, a positive correlation between sTNFR-1 and leptin was found in obese healthy subjects, after control for body fat mass (44). This may reflect different metabolic roles of the TNF- system in obesity and in infection. Adipose tissue itself is a site of production of both TNF- and sTNFR-1, most probably for purposes other than defense against infection, and such production may become apparent in the peripheral blood of obese persons (45).
Some limitations of this study should be noted. Plasma concentrations may not always reflect the biologic activity of compounds such as leptin and cytokines, in which diurnal rhythms and pulsatile release may occur (46) and the interaction with circulating receptors may be important (42, 47). Food intake was measured with the use of a 24-h recall protocol and a food-frequency questionnaire, methods that are often biased by over- or underreporting (48). Such limitations are shared with other studies on this subject (12, 22–24). However, this study has the benefit of systematic direct assessment of body composition by a validated technique. Our observations show that data on leptin concentrations in clinical human studies may be misleading if fat mass and energy metabolism are not measured concomitantly.
In conclusion, leptin does not appear to be part of the proinflammatory cytokine response in human pulmonary tuberculosis. Changes in leptin are entirely appropriate for the changes in body fat mass and energy balance. Altered leptin activity cannot, therefore, be held responsible for the weight loss and anorexia so often associated with tuberculosis infection.
ACKNOWLEDGMENTS
We are grateful to the nurses and physicians in the departments of Infectious Diseases and Chest Medicine, St George’s Hospital, and of Chest Medicine, St Helier Hospital (N Cooke), who were of great help in recruiting patients to this study. We are also grateful to all patients who contributed their time and effort to the study.
REFERENCES
1 From the Vanderbilt Center for Human Nutrition, Nashville, TN.
2 Reprints not available. Address correspondence to GL Jensen, Vanderbilt University Medical Center, 514 Medical Arts, Nashville, TN 37212. E-mail: gordon.jensen{at}mcmail.vanderbilt.edu.
See corresponding article on page1480.
Loss of body cell mass is a common and serious problem for patients with end-stage chronic obstructive pulmonary disease (COPD), especially those with emphysema. COPD patients with emphysema have lower body mass indexes and greater depletion of lean body mass than do COPD patients with chronic bronchitis (1). Nonetheless, skeletal muscle weakness is associated with wasting of extremity fat-free mass (FFM) in COPD patients, independent of airflow obstruction and COPD subtype (2). Indeed, body weight and body mass index are independent risk factors for mortality in COPD patients (3, 4). Not only are COPD patients often malnourished on hospital admission but many are subject to further decline during hospitalization.
Poor nutritional status in COPD patients has been related to adverse effects that may contribute to complications and increased mortality. Patients with low body weights have greater gas trapping, lower diffusing capacity, and less exercise capacity than do persons with similar respiratory mechanics but normal body weights. Loss of body cell mass is associated with a reduction in the mass of the diaphragm and of the respiratory muscles, resulting in declines in strength and endurance. A malnutrition-related decline in immune status may further blunt airway defenses. These effects can contribute to undesirable clinical sequelae that include hypercapnic respiratory failure, difficulty with weaning from mechanical ventilation, and nosocomial lung infections.
Although astute clinicians have long recognized that weight loss portends an ominous prognosis for patients with COPD, there has been little investigation of the underlying mechanisms of malnutrition in this setting. A variety of contributing factors have been proposed and it is likely that more than one factor is often at play. Disturbances in energy balance may reflect both the mechanical inefficiency of breathing and the reduced dietary energy intakes of these patients. In COPD patients, resting energy expenditure (REE) has been reported to be 15–20% above predicted values and the increased energy required for breathing has been suspected to account for the difference. Under controlled chamber conditions, the basal metabolic rate was found to be elevated in patients with stable COPD even though the daily total energy expenditure (TEE) was normal (5). It appeared as though the patients compensated by reducing their level of spontaneous physical activity and related energy expenditure. In contrast, independent of resting metabolic rate, the TEE was elevated in COPD patients as measured by the doubly labeled water method (6).
Complex changes in metabolism are ultimately the result of inflammation, hypoxia, hypercapnia, nutritional deprivation, and pharmacologic therapy. Stressors like nosocomial infection may exacerbate the situation by promoting hypermetabolism. Muscle proteolysis in the setting of systemic inflammatory responses appears likely in deteriorating patients. The ubiquitin-proteasome pathway is activated in catabolic states to accelerate the breakdown of muscle proteins. Cytokine-mediated cachexia, similar to other end-stage organ failure syndromes, is possible in COPD patients. Elevated concentrations of soluble tumor necrosis factor receptors and acute phase proteins have been observed (7) and anorexia and decreased dietary intakes are common. Steroid therapy may further stimulate proteolysis and promote gluconeogenesis through inhibition of both protein synthesis and the transport of amino acids into muscle.
One might think that there would be abundant data to support the efficacy of nutritional interventions for COPD patients but this is not the case. Although modest improvements in respiratory and limb muscle functions have been reported with nutritional repletion of ambulatory COPD patients, a recent meta-analysis of 9 randomized controlled intervention trials found that nutritional support had no effect on anthropometric measures, lung function, or functional exercise capacity among patients with stable COPD (8). A recent nutritional intervention trial found improvements in body weight and respiratory muscle strength in some COPD patients (3). Weight gain was a significant predictor of survival, but there were many nonresponders. Another study characterized the nonresponse to nutritional intervention among COPD patients as being related to aging, anorexia, and systemic inflammatory response (9).
In this issue of the Journal, Engelen et al (10) present an informative study of amino acid profiles in the quadriceps femoris muscle and arterial plasma of patients with COPD (n = 28) and of healthy, age-matched control subjects (n = 28). Of particular interest is the differentiation between the COPD patients with (Emph+; n = 14) and without (Emph-; n = 14) emphysema on the basis of macroscopic evidence of lung parenchyma destruction by high-resolution computed tomography. Low concentrations of plasma branched-chain amino acids were observed in COPD patients, which was attributed to a decline in leucine. The muscle-to-plasma leucine gradient was also elevated in COPD patients, as were plasma insulin concentrations. Additionally, concentrations of most amino acids in muscle were lower in the EMPH+ group than in the EMPH- group. Muscle glutamine was higher in EMPH- patients than in either the EMPH+ or the control subjects. REE measured by open-circuit indirect calorimetry, FFM measured by dual-energy X-ray absorptiometry, and REE:FFM were lower in the EMPH+ than in the EMPH- patients. Engelen et al suggested that alterations in leucine metabolism occur in patients with COPD and that there were striking differences in muscle amino acids between those with and without parenchymal destruction.
These findings indicate the need to carefully characterize the subtype of COPD when studying amino acid metabolism. Variation in the degree of parenchymal destruction may account for previously conflicting findings. Patients with severe parenchymal destruction tend to be the most malnourished and the least responsive to nutritional intervention. It is likely that COPD patients with macroscopic emphysema manifest the sequelae of both the inflammatory process and semistarvation. The result may be a combination of alterations in intermediary metabolism and negative energy balance that culminate in loss of body cell mass.
Engelen et al appropriately highlight the need for further investigation of several key observations.
Better understanding of the mechanisms leading to malnutrition in COPD patients should guide the development of improved interventions and help clinicians learn who should be targeted. By the time weight loss occurs in patients with end-stage emphysema, there is often little benefit of currently available nutritional support modalities. Can subjects at risk for malnutrition be identified early in the course of inflammatory responses before loss of body cell mass occurs? There is a need to develop novel interventions to augment nutritional support. Possible interventions include the use of anticytokines to blunt inflammatory responses and trophic agents to facilitate the accrual of muscle mass.
REFERENCES
1 From the Departments of Pulmonology and Physiology, Maastricht University, Maastricht, Netherlands.
2 Supported by a grant from the Netherlands Asthma Foundation (project no. 96.16). 3 Address reprint requests to HR Gosker, Department of Pulmonology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, Netherlands. E-mail: h.gosker{at}pul.unimaas.nl.
ABSTRACT
Low exercise tolerance has a large influence on health status in chronic obstructive pulmonary disease and chronic heart failure. In addition to primary organ dysfunction, impaired skeletal muscle performance is a strong predictor of low exercise capacity. There are striking similarities between both disorders with respect to the muscular alterations underlying the impairment. However, different alterations occur in different muscle types. Histologic and metabolic data show that peripheral muscles undergo a shift from oxidative to glycolytic energy metabolism, whereas the opposite is observed in the diaphragm. These findings are in line with the notion that peripheral and diaphragm muscle are limited mainly by endurance and strength capacity, respectively. In both diseases, muscular impairment is multifactorially determined; hypoxia, oxidative stress, disuse, medication, nutritional depletion, and systemic inflammation may contribute to the observed muscle abnormalities and each factor has its own potential for innovative treatment approaches.
Key Words: Chronic obstructive pulmonary disease • COPD • chronic heart failure • CHF • skeletal muscle • peripheral muscle • respiratory muscle • exercise intolerance • muscle performance • muscle morphology • muscle metabolism • hypoxia • oxidative stress • medication • disuse • nutritional depletion • systemic inflammation • oxygen therapy • antioxidant status • training • nutritional support • anabolic steroids • review
INTRODUCTION
According to the definitions of the World Health Organization, chronic diseases are characterized not only by the primary impairments they cause, but also by the disabilities or even handicaps that result from them (1). Although the primary impairments in chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF) clearly differ, there is a striking resemblance in the systemic consequences of these diseases and their effects on exercise capacity and health status (Figure 1). Impaired skeletal muscle function in COPD and CHF has long been ignored by focusing on the respective ventilatory and cardiac limitations on exercise performance. Research has shown that impaired skeletal muscle function is also an important predictor of exercise limitation in both diseases (2–6). Progression of the primary impairments in these disorders can be slowed down with medication (7, 8). Reversion can be only partially achieved through surgical interventions such as lung volume reduction surgery and lung transplantation (9, 10) and coronary bypass surgery and heart transplantation (11). However, there are limits on the age of most eligible patients and the availability of donor organs for these interventions. In addition, such interventions do not always confer a survival benefit; no improvement was found after lung transplantation in patients with end-stage emphysema (12). Also, irrespective of the reversibility of the organ impairment, exercise intolerance in both COPD and CHF remains after surgical intervention (13, 14), indicating that more detailed insight into the systemic consequences is required for effective treatment of these diseases.
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FIGURE 1. . Contributors to exercise intolerance in chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF).
Muscle function depends, though not completely, on perfusion, muscle mass, fiber composition, and energy metabolism (15). It can be inferred that alterations in one or more of these determinants play a role in reduced muscle performance. Indeed, such changes have been found in both COPD and CHF and there are striking similarities between the 2 etiologically distinct disorders.
In this review, we first present an overview of the clinical studies that have investigated impaired muscle function, with special emphasis on muscle morphology and energy metabolism in COPD and CHF. The advantage of discussing both diseases simultaneously is that the evidence about each complements that of the other and therefore provides more insight into the possible underlying causes of the muscle alterations. In the second part of the article, potential causes will be discussed, including hypoxia, oxidative stress, disuse, medication, nutritional depletion, and systemic inflammation. The third part deals with therapeutic perspectives.
MUSCLE ALTERATIONS IN COPD AND CHF
Muscle performance
Muscle performance is characterized largely by strength and endurance. Strength is defined as the capacity of the muscle to develop maximal force, and endurance is defined as the capacity of the muscle to maintain a certain force over time, thus, to resist fatigue. Loss of either one of these aspects results in muscle weakness and impaired muscle performance. Numerous studies have now convincingly shown that COPD and CHF are commonly associated with muscle weakness (6, 16–21). Probably the most extensive study on the influence of muscle weakness on exercise capacity in cardiorespiratory disorders was done by Hamilton et al (4). Compared with healthy subjects, patients with respiratory failure, heart failure, or a combination of both had significantly less strength in both peripheral and respiratory muscles. However, strength and endurance seem not to be affected in the same way in respiratory and peripheral muscles. This is illustrated by the poor correlation between the strengths of both muscle groups in the 2 disorders (18, 20, 21) compared with the much stronger correlation in healthy subjects (22). This implies that the strength component of muscle weakness is affected differently in peripheral and respiratory muscles. In healthy subjects as well as in patients with COPD or CHF, exercise-limiting symptoms are the sense of leg effort (exertional discomfort) or breathlessness (exertional dyspnea) (23, 24). Thus, despite correlations between peripheral muscle strength and performance in COPD and CHF (18, 23, 25), reduced endurance (ie, fatigue) seems to be the dominant limiting factor in peripheral muscles in these patients because the sense of leg effort was one of the main reasons to stop exercising (4, 24, 26–29). It was shown that early lactic acidosis occurs in COPD during exercise (30, 31) and that this is largely the result of lactate release from the lower exercising limbs (32). Muscle acidosis is a contributing factor to muscle fatigue (33).
Fatigue is probably not the main limiting factor in respiratory muscle function. Morrison et al (34) found that COPD patients have low respiratory muscle strength and endurance. Fatigue of the respiratory muscles may indeed occur during exercise, but it is not certain whether this is an independent determinant of exercise capacity (27, 35–38). In addition, it is unlikely that the respiratory muscles of exercising COPD patients contribute to the lactate response mentioned earlier (39). It should also be emphasized that the respiratory muscles must operate against the mechanical airway impedances in this specific disorder (40), for which the force component of respiratory muscle function is most likely of great importance. For CHF it was found that respiratory muscle strength and not respiratory muscle fatigability correlated with the degree of dyspnea (41). Thus, it seems that strength is the limiting aspect of muscle performance in the respiratory muscles, whereas endurance is limiting in peripheral muscles. However, more detailed studies are required to clarify the individual roles of strength and endurance limitation in peripheral and respiratory muscles in COPD and CHF.
Muscle morphology
In both CHF (23, 42–44) and COPD (3, 45–50), marked loss of muscle mass or decline in cross-sectional muscle area is observed. This muscle wasting plays an important role in the loss of exercise tolerance in these patients. However, morphologic alterations may also be related to impairment of muscle function, although direct relations with exercise performance have not yet been shown. Some histologic information is available on abnormalities in skeletal muscle in CHF but there is hardly any on COPD. Gertz et al (51) found no signs of increased fibrosis or other alterations in intercostal muscles of patients with respiratory failure, whereas endomysial fibrosis has been found in skeletal muscle of a limited number of CHF patients (52). Increased activity of acid phosphatase, a lysosomal enzyme contributing to protein degradation, has been found in the quadriceps of some patients with CHF (25) or respiratory failure (50). Increased lipid deposits have been found in the quadriceps, biceps, and deltoids of some patients with CHF (25, 52). Contradictory results have been obtained with respect to capillary density in peripheral skeletal muscle in CHF. A normal capillary density has been found (25), which agrees with results of 2 other studies in which both reduced capillary-fiber ratios and atrophy resulted in unchanged capillary densities (53, 54). An unaltered capillary-fiber ratio has also been reported, however, with greater capillary density due to fiber atrophy (55). In contrast, reduced capillary density in combination with a reduced capillary-fiber ratio has been shown in CHF patients (56) and even in heart transplant recipients (57). Thus, overall, there is a tendency toward a reduced capillary-fiber ratio, but depending on the degree of atrophy, the capillary density may even be elevated. This tendency was recently confirmed in COPD (46).
In a few studies, morphometry of mitochondria with use of electron microscopy showed that mitochondrial volume densities in skeletal muscle were lower in CHF patients than in control subjects (56, 58), and this was still the case 10 mo after heart transplantation (57). Histochemical alterations reflecting mitochondrial abnormalities have also been reported in biceps muscle biopsies of COPD patients (50). These results suggest that the oxidative capacity of peripheral skeletal muscles may be altered in both diseases.
Muscle fiber type distribution
Probably the most remarkable muscle alteration in COPD and CHF is a relative shift in fiber composition that seems to occur in opposite directions in peripheral and respiratory muscles. Fiber typing is mainly performed histochemically, and is based on differences between fibers in myosin ATPase activities or immunocytochemistry (59). Adult mammalian skeletal muscle contains 4 myosin heavy chain (MyHC) isoforms, namely, types I, IIa, IIb, and IIx (60). In most older studies, fiber typing was limited to determining fiber types I, IIa, and IIb. Furthermore, human fibers formerly identified as being IIb with myosin ATPase staining are probably IIx fibers (61). Therefore, the notation IIb/x is used in the subsequent text. Fiber type I has a slow twitch and develops a relatively small tension, but because it depends mainly on aerobic metabolism, it is fatigue resistant. In contrast, fiber type IIb/x has a fast twitch and develops large tensions, but it is susceptible to fatigue because its energy conversion is based on anaerobic, glycolytic metabolism. Fiber type IIa has intermediate properties in that it also has a fast twitch, develops a moderate tension, is relatively resistant to fatigue, and is apt to work under both aerobic and anaerobic conditions (15, 59, 62).
A lower percentage of type I fibers and a corresponding higher percentage of type II (mainly type IIb/x) fibers, compared with those of normal subjects, has been reported in limb muscles of COPD (46, 63–66) and CHF (25, 53–56, 67) patients. In addition, in one of these studies an increase in intermediate fiber types (I + II) was also observed in CHF patients (55). These fibers may represent transformation intermediates in the IIIb/x shift. In contrast with that in peripheral muscles, a shift from type IIb/x to type I fibers has been reported in the diaphragms of both COPD and CHF patients. In healthy subjects, the diaphragm has 50% type I, 25% type IIa, and 25% IIb/x fibers (68), whereas the diaphragms of CHF patients contain 60% type I, 35% type IIa, and only 10% type IIb/x fibers (69). A IIb/xI shift was also observed when the distribution of MyHC isoforms was analyzed in diaphragms of patients with CHF (70) or COPD (71). Furthermore, a larger population of type I fibers in the diaphragm (corrected for the percentage of type I fibers in the quadriceps femoris) was found in both COPD and CHF patients (26, 72) compared with sedentary control subjects (68, 73). The proportion of type I fibers in both the internal and external intercostal muscles was 62% in both COPD patients and control subjects in some studies (68, 74), but in other studies COPD patients had lower proportions of type I fibers (46–48%) in these muscles (73, 75). Also, elevated expression of fast MyHC has been reported in the external intercostal muscles of COPD patients (76). These results suggest that the accessory respiratory muscles do not show the III fiber shift that occurs in the diaphragm. No such data have been published for CHF patients.
The overall outcome of the studies done until now (despite some variation in the results) has been that there is a IIIb/x shift in peripheral muscles and a IIb/xI shift in the diaphragm in COPD and CHF. It is possible that these shifts have functional consequences in the affected muscles because the distinct fiber types have different contractile properties with respect to twitch and fatigue resistance. Therefore, in COPD and CHF, a IIIb/x shift accompanied by more glycolytic and less oxidative capacity in peripheral muscles implies loss of fatigue resistance. This change might contribute to the observed loss of exercise tolerance because peripheral muscle fatigue is the main exercise-limiting factor in these patients. This was confirmed by a study in which a faster twitch response in combination with less resistance to fatigue was observed in the leg muscles of CHF patients (77). Accordingly, a IIb/xI shift toward more oxidative metabolism in the diaphragm implies a shift toward a more fatigue-resistant but less strength-adapted muscle. This too is in line with our notion that strength and not fatigue seems to be the main limiting factor for respiratory muscle function.
Muscle metabolism
Much data are available on skeletal muscle metabolism in CHF and COPD, partly because of the applicability of 31P-nuclear magnetic resonance (31P-NMR), which has enabled a direct and noninvasive assessment of tissue concentrations of high-energy phosphates and pH. High concentrations of ATP, creatine phosphate (CrP), and nicotinamide adenine dinucleotide in the reduced form (NADH) reflect a high-energy state, whereas elevated concentrations of ADP, AMP, inorganic phosphate (Pi), and oxidized nicotinamide adenine dinucleotide (NAD+) commonly reflect a low-energy state. Lactate and glycogen concentrations are often measured in muscle metabolism, but note that low concentrations may reflect either increased clearance or reduced formation, and vice versa for high concentrations. Although activities of enzymes involved in muscle energy metabolism measured in vitro do not reflect the physiologic situation because maximal activities are obtained under optimal, artificial circumstances, they do provide an indication of adaptations in the expression of proteins involved in metabolic pathways. Typical oxidative enzymes are citrate synthase, succinate dehydrogenase, and ß-hydroxyacyl-CoA dehydrogenase (HAD). Typical glycolytic enzymes are hexokinase, phosphofructokinase, and lactate dehydrogenase—the latter of which catalyzes the last step of anaerobic glycolysis.
Measurements of substrate and cofactor concentrations in peripheral skeletal muscle of COPD and CHF patients indicate impaired energy metabolism (Table 1). Most striking are the observed reduced concentrations of high-energy phosphates at rest. Pouw et al (81) observed the higher Pi-CrP and ADP-ATP ratios were associated with slightly but statistically significantly elevated inosine monophosphate (IMP) concentrations. The latter may be due to increased degradation of accumulating AMP by deamination, which probably reflects reduced aerobic capacity (83). The situation becomes even worse during exercise: a greater increase in the Pi-CrP ratio and a faster drop in pH were found in the calf muscle of COPD (47, 84, 85) and CHF (55, 86, 87) patients performing exercise than in healthy persons. Similar results were obtained for the forearm muscle (14, 87–89). In addition, a slower recovery of CrP concentrations was observed after exercise in COPD and CHF patients than in healthy persons (14, 47, 55, 85–89). These results suggest that rephosphorylation of high-energy phosphates is less efficient in these patients, both during and after muscular exercise. In addition, glycogen contents in COPD and CHF patients tend to be lower, whereas lactate concentrations are higher than in healthy persons (Table 1). Thus, it seems that anaerobic energy metabolism is enhanced in these diseases, and because this process yields far less ATP than does complete oxidative degradation of glucose, this could explain the reduced high-energy phosphate concentrations.
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TABLE 1. . Muscle metabolite concentrations in chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF)1
Analysis of enzyme activities also suggests an overall increase in glycolytic and an overall decrease in oxidative activities in peripheral muscles of both COPD and CHF patients (Table 2). Because these enzyme activities depend largely on the fiber type (95), it is likely that this shift in activities is related to the shift in fiber distribution mentioned above. Whether enzyme activities adapt to the fiber type redistribution or the fiber type redistribution adapts to enzyme activities remains unclear. In addition to these chronic alterations of enzyme activities, which are measured in vitro, there is probably also an acute effect on the activities of these enzymes. As a consequence of impaired electron transport, regeneration of NAD+ from NADH is reduced and citrate synthase and HAD are inhibited by a high NADH-NAD+ ratio (96). In addition, elevated AMP concentrations, resulting from the inefficient rephosphorylation of ATP, stimulate glycolysis (96). However, note that this acute effect is invisible in the in vitro activity measurements. In 2 studies, an additional inverse relation of oxidative enzyme activities with arterial lactate concentrations was found during exercise, emphasizing the assumed shift from oxidative toward glycolytic energy generation (30, 91). This loss of oxidative capacity probably accounts for the above-mentioned lipid deposits (97) because fatty acid consumption may be reduced while the supply of blood fatty acids continues. Recently, activities of 2 other oxidative enzymes, cytochrome-c oxidase (COX) and NADH dehydrogenase, were found to be elevated in COPD and CHF patients (Table 2). On first notice, this seems to be paradoxical in light of the observed reductions in the oxidative enzymes mentioned earlier. However, the oxidative enzymes mentioned earlier are involved in either the citric acid cycle or fatty acid oxidation, whereas COX and NADH dehydrogenase are enzymes of the respiratory chain. COX interacts with oxygen and therefore is the main determinant of mitochondrial oxygen affinity (98). Because a correlation between COX activity and hypoxemia was found (93), it may be that an increased number of COX molecules is a mechanism that enhances the efficiency of residual oxygen extraction and utilization and, thus, respiratory chain function. In this study, COX-I (a mitochondrial encoded subunit of COX) messenger RNA concentrations were not elevated but mitochondrial 12S ribosomal RNA concentrations were. Because this particular ribosomal RNA is a component of mitochondrial ribosomes, which are involved in translation, mitochondrial protein synthesis may be enhanced. The mechanism of hypoxia sensing and subsequent stimulation of mitochondrial gene expression remains, however, unclear (93).
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TABLE 2. . Muscle enzyme activities in chronic obstructive pulmonary disease (COPD) and chronic heart failure (CHF)1
Because of technical difficulties with 31P-NMR and muscle biopsies of the diaphragm and accessory respiratory muscles, little is known about energy metabolism in these muscles. However, the observed alterations in enzyme activities (Table 2) confirm the morphologic data, in that oxidative enzyme activities are reduced and glycolytic enzyme activities are elevated in COPD and CHF compared with the healthy state. As in peripheral muscles, this shift probably results from the shift in fiber type distribution.
POSSIBLE UNDERLYING FACTORS
Hypoxia
In COPD and CHF, oxygen delivery to peripheral and respiratory muscles may be insufficient as a result of hypoxemia, reduced blood supply, or both. In both cases, muscle tissue may become hypoxic and this could lead to the adaptive changes in skeletal muscle described above. In this respect, relevant information is now available from mountaineering expeditions (lasting 6 wk above 5000 m), because oxygen is limited at this altitude. Under these conditions, reductions in mitochondrial volume densities, oxidative enzyme activities, and cross-sectional areas of muscle fibers were found in the quadriceps (99–101). Note, however, that such expeditions are accompanied by strenuous physical activity, which also causes muscular adaptations other than those caused by hypoxia. In fact, the effect of training in combination with hypoxia may even cause a shift toward more oxidative metabolism (102). More information about the effect of hypoxia on muscle has been obtained from animal studies.
Several animal studies have shown that hypoxia can indeed lead to the muscular alterations described for the limb muscles of COPD and CHF patients. For example, reduced fiber diameters in combination with unaffected numbers of capillaries, resulting in increased capillary densities, have been reported in rats exposed to hypoxia (103–105). It is suggested that the availability of oxygen to remaining muscle mitochondria is enhanced by this increased capillary density in combination with the loss of oxidative capacity (99). Furthermore, animal studies showed that hypoxia depresses protein synthesis (106–109), even in muscle tissue (106, 107). Whether hypoxia itself can contribute to the shift in muscle fiber distribution observed in COPD and CHF patients remains uncertain. There is evidence that chronic hypoxia inhibits the normal conversion of type IIa to type I fibers in growing rats, with the final outcome that these rats have a predominating proportion of type IIa fibers, unlike in control rats (110–112). However, no differences in fiber types were found when full-grown, adult rats were exposed to chronic hypoxia (110, 111, 113). Thus, it seems that hypoxia does not directly cause a type III fiber shift and it is more likely that the abnormal fiber type distribution results from alterations in muscular development. A similar mechanism may underlie the abnormal fiber type distribution in the regeneration of damaged muscle or the adaption of muscles to consequences of the disease in COPD and CHF.
In addition, there is evidence that hypoxia causes a shift toward glycolytic metabolism. In studies in which rats were exposed to intermittent hypoxia, it was found that citric acid cycle activity was reduced whereas glycolytic metabolism was enhanced, resulting in an increased ratio of lactate to pyruvate (114, 115). Malate dehydrogenase, a citric acid cycle enzyme, was also found to be reduced by hypoxia (116). Furthermore, hypoxia causes stimulation of glucose transport (117) and increased concentrations of membrane-associated glucose transporters (GLUT1 and GLUT4) in rat muscle (118). In muscle cell cultures, this up-regulation of GLUT1 can be mediated by either hypoxia or inhibition of the respiratory chain (119), suggesting that hypoxia affects glucose transport (and probably also metabolism) via impairment of oxidative phosphorylation.
However, in COPD and CHF this reduction in oxidative capacity does not occur in the diaphragm. Hypoxia may cause an endurance training effect in the diaphragm because of increased ventilation, which overrides its direct effect, ultimately resulting in a shift toward more aerobic metabolism.
Oxidative stress
Oxidative stress may be another factor contributing to muscle damage via reactive oxygen species. Increased plasma concentrations of lipid peroxidation products have been found in both COPD and CHF patients (120, 121). The main source of these oxygen free radicals is mitochondria because 2–5% of the oxygen consumed is not fully reduced in the electron transport chain and may leak away as superoxide radicals (122, 123). An alternative source of free radicals is immune cells activated during inflammation (124). Monocytes and macrophages produce the cytokine tumor necrosis factor (TNF-), which may in turn induce oxidative stress in myocytes (125). Indeed, elevated TNF- blood concentrations have been found in both COPD (126–128) and CHF (129–133) patients, particularly in those patients with weight loss or muscle wasting. A third generator of free radicals is xanthine oxidase, which is involved in the deamination of AMP to IMP under conditions of very high AMP concentrations, such as a low-energy state (123). The above-mentioned elevated IMP concentrations in COPD (81) suggest enhanced AMP breakdown.
Susceptibility to these free radicals depends largely on the antioxidant status of tissues (123). The main antioxidant scavengers and enzymes are, among others, reduced glutathione, vitamin E (in cell membranes), superoxide dismutase, glutathione peroxidase, and catalase (123, 134, 135). Repeated exposure to oxidative stress, during long-term physical training for example, stimulates the defense system against oxygen free radicals in that concentrations of scavengers and activities of antioxidant enzymes increase (122, 123, 134–136). Oxygen flux to muscles and the resulting oxidative stress can increase tremendously during exercise (123, 137) and the disuse of muscles thus may take away this antioxidant-stimulating trigger and result in low antioxidant status. Chronic hypoxia probably acts in the same way because less oxygen is available to form reactive oxygen species. Limitations of oxygen supply are indeed found to be associated with reductions in superoxide dismutase activity in mammalian tissues like brain, lungs, and heart, although this change was not found in skeletal muscle tissue (138, 139). In addition, in myocytes obtained from chronically hypoxic human myocardium cultured at low oxygen tension, antioxidant enzyme activities were lower than in myocytes cultured at a higher oxygen tension, illustrating the direct modulatory effect of oxygen (140). In vivo and in vitro hypoxia-reoxygenation studies showed that oxygen oversupply after a period of oxygen shortage may give rise to free radical formation in myocytes (138, 141, 142). Accordingly, in COPD and CHF patients, chronic hypoxia may result in reduced antioxidant status and occasional bouts of exercise may cause a burst of free radicals that exceeds the capacity of the defense system (122). It is also possible that the patients' reduced oxidative capacity itself leads to enhanced oxidative stress because the sudden oversupply of oxygen during exercise is inefficiently metabolized.
Reactive oxygen species are capable of damaging lipids and proteins (122, 123, 134, 143). Radicals that react with fatty acyl moieties in membrane phospholipids cause a chain reaction of peroxidations that increase the membrane permeability (143). Maintenance of membrane integrity is crucial for adequate functioning of the respiratory chain because the driving force for oxidative ATP synthesis is the electrochemical proton gradient over the inner membrane of the mitochondrion, which is generated during the electron transfer from NADH to oxygen (96). Leakage of ions through a more permeable mitochondrial inner membrane may thus impair mitochondrial function by uncoupling oxidative phosphorylation. Indeed, rats with an inherited overproduction of oxygen free radicals showed a higher degree of lipid peroxidation and protein damage in combination with impaired respiratory chain function in liver mitochondria than did control rats (144). Furthermore, a marked decrease in ATP concentrations was observed in cultured endothelial cells exposed to reactive oxygen species (145). In addition, there is evidence that an intracellular calcium overload, probably caused by a damaged sarcoplasmic reticulum membrane in combination with impaired activity of calcium ATPases, accompanies oxidative stress in animal myocytes (122, 138, 141, 142, 146–148), which may further uncouple respiration from ATP production through extensive depolarization of the inner membrane (149).
Protein oxidation by oxygen free radicals leads to formation of carbonyl groups on amino acid residues, which may modify the structure or chemical properties of the proteins affected (150). These alterations may cause a decline in protein function or even complete protein unfolding. The latter gives rise to enhanced susceptibility to proteinases. These modified proteins may also be recognized as foreign substances and, hence, be attacked by the immune system. Whether radical-induced protein damage plays a role in the abnormalities in muscles of COPD and CHF patients is unclear. It was shown in animal studies that oxidative stress induced in vivo caused myofibrillar muscle protein modification and that these proteins were rapidly degraded by proteases (151). Thus, theoretically, muscle atrophy can be enhanced by radical-induced protein damage. Indeed, it was shown that a calcium overload is involved in muscle atrophy (152) and that vitamin E deficiency facilitates muscle wasting and necrosis (153), both probably mediated by oxidative damage to proteins. Also, in human skeletal muscle it was shown that mitochondria and mitochondrial proteins were more susceptible to oxidative damage than were other subcellular components (154), which suggests that protein damage may cause impaired oxidative metabolism.
As opposed to necrosis, which is the result of exogenous damage as described above, apoptosis of muscle cells is an active process of cell death, which has also been associated with oxidative stress (155). In this study, the exposure of rat myoblasts to nitric oxide or hydrogen peroxide led to apoptotic cell death. Because these chemical stimuli are also released by immune cells, we cannot exclude the possibility that apoptosis underlies muscle wasting during inflammation.
Disuse
Disuse of skeletal muscle from a low level of physical activity is also a factor that most likely contributes to the observed muscle alterations in COPD and CHF. Hampered by their disease, these patients perform less physical activity, which may have a detraining effect on their peripheral muscles. First, detraining causes muscle weakness because of reduced motor neuron activity and muscle wasting (59, 156). Second, disuse may cause a relative reduction in the percentage of type I fibers and an increase in the percentage of type IIb/x fibers (59, 157). Third, detraining causes a decline in the activity of enzymes involved in oxidative energy conversion. This occurs in both type I and type II fibers (157, 158), suggesting that loss of oxidative capacity can occur even without any change in fiber composition. As mentioned earlier, muscular disuse has a negative effect on antioxidant status and, hence, enhances the risk of oxidative damage. Reduced physical activity thus contributes to the loss of muscle mass and probably also to the III fiber shift and the reduced oxidative capacity observed in limb muscles in COPD and CHF patients. As mentioned above, the diaphragm is probably not disused in these diseases and a kind of endurance training effect may even occur. This may be true not only for COPD but for CHF as well because in severe CHF, dyspnea and elevated ventilation occurs even at rest (28, 159).
Medication
Of the wide range of drugs used to treat COPD and CHF, only corticosteroids have been associated with skeletal muscle alterations. Especially in COPD, but less frequently in CHF, corticosteroids are used in low doses as maintenance medication or in higher doses during acute disease exacerbations (160, 161). Depending on the type of steroid, the dose, and the duration of treatment, the drug-induced defects range from alterations in energy metabolism to muscle weakness with underlying muscle wasting and histologic abnormalities (160, 161). From the results of both clinical and animal studies it became clear that steroid-induced muscle wasting is often associated with an overall negative nitrogen balance, reduced protein synthesis, increased protein catabolism in muscle tissues, and increased plasma concentrations of amino acids (162). These findings suggest that corticosteroids probably stimulate the mobilization of amino acids from muscle proteins (161), supplying the liver with gluconeogenic precursors, which corresponds with a shift toward glycolytic metabolism. No such data are available for CHF, but a few reports showed that COPD patients who chronically receive corticosteroids indeed may show more muscle weakness alone or with an accompanying loss of muscle mass than do nontreated COPD patients (17, 163–165). However, because COPD patients receive corticosteroids to treat inflammation, it is difficult to distinguish between the effect of steroid administration and the effects of the disease exacerbations. Also, decreased testosterone concentrations have been reported for male COPD patients receiving glucocorticoids (166).
Assuming that corticosteroids are involved in muscular alterations, the question arises as to whether this effect is generalized or is muscle-type specific. Experimental studies indicate that the glycolytic fibers seem especially susceptible to steroid-induced muscle wasting (161, 162, 167), suggesting that vulnerability depends on muscle fiber composition. The diaphragms of affected patients have a relatively high proportion of type I fibers, and so could therefore be less affected by steroid usage; limb muscles, consisting of more type II fibers, would be more vulnerable. On the other hand, the diaphragm already has a lower type II fiber content and selective wasting of type II fibers could further reduce diaphragm strength. Because strength is the main limiting factor in diaphragm muscle performance, it may be more vulnerable to corticosteroids than are limb muscles. Furthermore, it is difficult to isolate the effect of corticosteroids on diaphragm function from the complex of other unfavorable influences on lung function in COPD (165). Therefore, Wang et al (168) studied the effect of prednisone on the respiratory muscle function of normal subjects (who had no disturbing influences on function) and found no differences from a control group. This does not exclude the possibility that other steroids might have an effect because a recent animal study showed different effects of fluorinated and nonfluorinated steroids at equipotent doses (169). The results of other animal studies confirm the hypothesized shift toward glycolytic metabolism in peripheral muscle and the shift toward oxidative metabolism in the diaphragm: in corticosteroid-treated rats it was found that diaphragm force generation and the proportion of type IIb fibers was reduced in combination with decreased activity of the glycogenolytic enzyme phosphorylase and increased activities of HAD and citrate synthase (170). Increased phosphofructokinase and glycogen synthase activities have been reported for limb muscle of corticosteroid-treated rats (169). Expressed per gram of muscle, limb twitch tension may even increase in steroid-treated animals, which indicates an increase in type II fiber content (169, 171). The fact that corticosteroids selectively affect type II fibers but are also associated with a shift toward glycolytic metabolism in peripheral muscle seems contradictory and needs further investigation.
Note that in both CHF and COPD, muscle performance is not fully recovered 5–38 mo after heart or lung transplantation; intrinsic skeletal muscle alterations remain (57, 172). Corticosteroids and cyclosporin are often used as immunosuppressive agents after transplantations and it is therefore possible that these drugs might be involved in impaired muscle function (13, 57, 172).
Nutritional depletion and systemic inflammation
Nutritional depletion commonly occurs in COPD (173, 174) and CHF (42, 175). In both disorders, nutritional depletion is an important determinant of exercise capacity (3, 44, 45, 176). Body weight (usually corrected for height) is often used to determine the nutritional status of patients, but this method neglects the differences in body composition between individuals (3). Determination of body composition with respect to nutritional depletion is important because different patterns of weight loss can be distinguished: predominant loss of fat mass, predominant loss of fat-free mass, or a combination of both.
Predominant loss of fat mass involves an impaired balance between energy requirements and energy intakes. Dietary intake can be low in COPD because of symptoms such as dyspnea, fatigue, and early satiety (177). Recently, systemic inflammation was suggested to affect appetite and dietary intake, mediated by the appetite-regulating hormone leptin (178). However, patients with COPD may lose weight despite a normal or above-normal dietary intake (179). In COPD and CHF, resting energy expenditure (REE) is often elevated (180–183). In addition, total daily energy expenditure that is elevated independently of REE has been found in COPD patients (184). Increased oxygen cost of breathing probably contributes to the increased total daily energy expenditure due to increased hyperinflation during exercise (185, 186), but because hyperinflation is not increased at rest, it is unlikely that an elevated oxygen cost of breathing accounts for the elevated REE (180). Suggested contributors to the elevated REE are the thermogenic effects of bronchodilating agents (180) and systemic inflammation (127). Furthermore, the observed loss of efficient aerobic energy metabolism might play a role in the increased REE and total daily energy expenditure. In this situation of semistarvation, loss of both fat mass and fat-free mass occurs, but the fat-free mass is relatively preserved. Therefore, intrinsic muscle abnormalities besides muscle mass probably account for impaired muscle performance.
Studies of muscle function and histology in anorexia nervosa patients have provided strong data on the effect of undernutrition in muscles. Muscle performance is markedly impaired in these patients (187–189) and is associated with weight loss, loss of muscle mass, and fiber atrophy (particularly of type II fibers) (190, 191). Data from animal studies confirm these effects of undernutrition. Loss of muscle mass associated with fiber atrophy was observed in limb muscles during nutritional deprivation (171, 192). Activities of the oxidative enzymes succinate dehydrogenase and HAD were found to be reduced (192, 193). The activity of the glycolytic enzyme phosphofructokinase was also found to be reduced (193), but this was not confirmed by Koerts-de Lang et al (169). In addition, high ADP and low CrP concentrations were observed in food-deprived animals (193, 194), suggesting that muscle energy metabolism is indeed impaired after deprivation. However, it remains unclear whether nutritional deprivation results in a general loss of activities of enzymes involved in energy metabolism or predominantly affects either oxidative or glycolytic energy metabolism. The contribution of nutritional depletion to a shift from oxidative to glycolytic metabolism in COPD and CHF patients needs further investigation.
Predominant loss of fat-free mass involves an impaired balance between protein anabolism and catabolism that results in the loss of fat-free mass. In emphysema, reduced muscle protein synthesis was found (49), but protein degradation was probably not increased (195, 196). Also, nitrogen intake was not low in these patients but nitrogen excretion was elevated (196). Amino acids may be required in processes other than muscular protein synthesis, such as gluconeogenesis. Because weight loss and loss of fat-free mass have often been associated with systemic inflammation in both COPD and CHF (126, 127, 133), it is also possible that amino acids are required for increased synthesis of inflammatory proteins in the liver. Disturbed plasma and muscle amino acid concentrations have been observed in COPD, suggesting that amino acids are indeed redirected from muscle (197). Animal and in vitro studies confirm this notion (198): exposure of myocytes to TNF- resulted in a decrease in protein synthesis, which occurred even during anabolic stimulation with insulin-like growth factor I (IGF-I). Furthermore, chronic administration of TNF- led to increased muscle protein catabolism and liver protein anabolism in rats. In addition, the above-mentioned involvement of TNF- in oxidative stress may contribute to muscle wasting (125). Protein depletion itself may impair skeletal muscle performance as reflected by reduced maximum voluntary handgrip strength, reduced respiratory muscle strength, and increased fatigability of in vivo electrically stimulated adductor pollicis muscle (199).
THERAPEUTIC PERSPECTIVES
Training
It is obvious that exercise training improves muscular performance because, depending on the training program, strength, endurance, or both improve (59). Because disuse has been suggested to be an important factor responsible for the alterations in muscle metabolism in COPD and CHF, it is possible that training the affected muscles could reverse these abnormalities. Indeed, exercise training improves exercise capacity in both COPD (200, 201) and CHF (6, 43, 202) patients. Furthermore, increased cross-sectional areas of oxidative fibers and elevated oxidative enzyme activities in the quadriceps muscle in combination with less arterial lactate accumulation during exercise have been found in trained COPD patients (46, 203). Training-induced increases in oxidative capacity and muscle mass of the quadriceps muscle have also been reported for CHF patients (58, 87). The above-mentioned exercise-induced increase in the Pi-CrP ratio and drop in pH in muscle is less after training (202). Thus, in peripheral muscles, training induces a partial improvement of the oxidative capacity in combination with increased exercise performance. In general, prolonged endurance training leads to increased percentages of type I and IIa fibers accompanied with greater oxidative capacity, resulting in higher fatigue resistance (59). Therefore, considering that fatigue is the main limiting factor in peripheral muscle performance, an endurance training protocol may be most suitable for improving the exercise capacity of limb muscles in COPD patients. This is also illustrated by the fact that in COPD patients, quadriceps endurance shows a larger improvement with training than does strength (200, 201).
No data on improvement of oxidative capacity with training are available for respiratory muscles. However, the differences between respiratory and peripheral muscles in COPD and CHF suggest that different training approaches are required to effectively improve their performances. Whereas respiratory muscle training in CHF remains an unexplored field, a variety of studies have been performed for COPD (204). Although training of respiratory muscle may improve its performance, there is little evidence of real clinical benefit. The best results are probably obtained with so-called resistance training (204), in which the inspiratory muscles are subjected to an increased pressure load. The fact that this is a kind of power training affecting respiratory muscle strength especially suggests that training of the diaphragm should be more focused on strength than on endurance (205, 206).
Another possible positive effect of exercise training is the increase in antioxidant status. As discussed above, disuse (or "disuse hypoxia") has a negative effect on antioxidant status and may therefore promote oxidative damage during occasional exercise because of the temporarily enhanced oxygen supply to the exercising muscles. However, regular physical exercise involves a regular increase in exposure of muscle tissue to oxygen and training thus probably reduces the risk of oxidative stress (135).
Nutritional support, anabolic steroids, and antiinflammatory therapy
The effects of nutritional support strategies on muscle mass and muscle function have been investigated in COPD, but it is a relatively unexplored area in CHF. Several studies in nutritionally depleted patients with COPD have shown that nutritional supplementation can improve both respiratory and peripheral muscle function (174, 207, 208). It is unclear, however, to what extent this improvement in muscle function is related to the increase in muscle mass per se (209). Muscle performance may reach normal values with nutritional support while muscle mass is still lower than that of control subjects, as shown for example in anorexia nervosa patients (189), which suggests that repletion of intrinsic muscle abnormalities is important in the improvement of muscle function. An early and a late response to nutritional supplementation has been proposed (199). After the first few days of repletion, muscle function improves 10–20% without any demonstrable gain in tissue protein. This early response probably results from improved electrolyte content (210) and improved concentrations of energy-rich compounds (51, 199). Only during prolonged treatment do physiologic functions further improve, accompanied by an increase in tissue protein and muscle mass (211).
However, a substantial subgroup of COPD patients did not gain weight in response to high-energy nutritional therapy (212). This subgroup was characterized by an elevated systemic inflammatory response, as evidenced by high concentrations of acute phase proteins and soluble TNF receptors. As mentioned earlier, systemic inflammation is associated with protein catabolism and probably plays a role in the loss of muscle mass. This suggests that antiinflammatory therapy might be beneficial in this particular subgroup. Many COPD patients receive inhaled or oral corticosteroids to treat local inflammation and acute infections. Systemic inflammation, however, is not reversed during this treatment (213). In addition, oral steroids may have a negative effect on skeletal muscle as mentioned above. A possible way to modulate systemic inflammation is through ingestion of polyunsaturated fatty acids (PUFAs). PUFAs are incorporated into the phospholipids of the cell membrane and play an important role in the regulation of inflammatory processes. Indeed, fish-oil supplementation reduced inflammatory mediators and had an anticachectic effect in pancreatic cancer patients (214). No studies are yet available regarding PUFA supplementation in COPD or CHF.
Administration of anabolic steroids may be an additional mode of intervention to counteract protein catabolism either by the androgen receptor–mediated promotion of protein anabolism or by neutralizing the effects of glucocorticosteroids through binding competition for the receptor mediating catabolism (215). Anabolic steroids could thus be useful in patients with muscle wasting, especially in those who are treated with corticosteroids. Anabolic steroid treatment in addition to nutritional support as an integrated part of a pulmonary rehabilitation program produced significantly enhanced fat-free mass despite a similar weight gain with nutritional support only. This increased fat-free mass was reflected in improved respiratory muscle function (209, 216). No difference in response was noted for patients receiving maintenance oral corticosteroid treatment (209). Currently, the effects of this combined treatment approach on peripheral skeletal muscle function, exercise performance, and health status is being studied. Besides effects on muscle performance, anabolic steroids resulted in an improvement in negative acute phase proteins such as albumin and transthyretin (215) in depleted COPD patients. This may indicate an antiinflammatory effect.
Others have investigated the effects of adjuvant treatment with recombinant human growth hormone (rhGH). Administration of this hormone induces lipolysis, protein anabolism, and muscle growth, either directly or through IGF-I. Two uncontrolled studies showed the effects of rhGH in nutritionally depleted patients with COPD. Administration of rhGH for 8 d (0.03 mg•kg-1•d-1 subcutaneous for 4 d, plus 0.06 mg•kg-1•d-1 for another 4 d) did not increase respiratory and peripheral skeletal muscle strength in COPD (217). In contrast, an increase in inspiratory muscle strength was reported after 3 wk of treatment (0.05 mg•kg-1•d-1 subcutaneous) (218). With use of a similar treatment regimen, but in a placebo-controlled fashion, the effects of administration of rhGH on body composition, resting metabolic rate, and functional capacity in underweight COPD patients in a stable clinical state were studied (219). Although fat-free mass increased significantly during the 3-wk treatment period, no improvement was seen in muscle function and exercise capacity even decreased in the treatment group. Furthermore, a significant increase in resting metabolic rate was observed.
In the previous sections of this article, we stated that COPD and CHF patients may have increased oxidative stress, in either muscle or lung tissue. Furthermore, vitamin E deficiency is associated with the pathogenesis of the wasting and weakness in thalassemia major (220). Therefore, another mode of nutritional intervention might be supplementation with antioxidants such as vitamins, glutathione, and N-acetylcysteine. Several studies indeed showed a beneficial effect on wasting of antioxidant supplementation (221). For example, vitamin E protects human skeletal muscle from damage during surgical ischemia-reperfusion (222) and vitamin C supplementation reduces exercise-induced oxidative stress (223). Similar results have been obtained in animal studies (153, 224, 225). Although antioxidant supplementation does reduce physical exercise–induced oxidative stress, it remains unclear whether exercise performance is enhanced (134, 221). Most of these data were obtained from athletes, who already have a high exercise capacity, whereas vitamin supplementation may have more effect in COPD and CHF patients, who have a very low exercise capacity. In addition, there are some indications that vitamin supplementation may improve lung function (226, 227). Antioxidant administration in CHF and COPD therefore deserves further investigation.
Oxygen therapy
Long-term oxygen therapy (LTOT) improves survival and quality of life of COPD patients (228, 229), but no such data are available for CHF. It is clear that acute oxygen administration is beneficial for exercise capacity in COPD (230–232). However, very little is known about the ability of LTOT to reverse the alterations found in skeletal muscles of COPD patients. In fact, improved exercise capacity during oxygen administration, including LTOT, could very well be an acute effect with no reversal of these abnormalities. First, by supplying oxygen, hypoxemia is partly reversed and with that dyspnea may be improved (230). The latter is an important determinant of exercise tolerance in COPD. Therefore, relief of breathlessness may account for a great deal of improvement in exercise capacity (231). Second, the acute supply of oxygen to muscle tissue probably improves oxidative energy metabolism only during the oxygen administration period itself, because the indexes of oxidative energy metabolism (Pi-CrP, pH, and CrP recovery) showed some improvement in a group of COPD patients only during oxygen administration (85). After exercise while breathing room air, COPD patients receiving LTOT still had a low Pi-CrP ratio and low pH in combination with slow CrP recovery compared with control subjects. Also, supplementation of oxygen does not add to the improving effects of training (232). Only in one study was there a reported improvement of the CrP-(CrP + Cr) ratio in resting muscle of COPD patients while breathing room air after 6–9 mo of LTOT (233). However, because the partial pressure of oxygen in blood also improved, this increase was probably caused by an increased oxygen supply and was not due to any reversal of muscle abnormalities. In addition, the low glycogen concentrations failed to improve, which further suggests that the muscle abnormalities were not reversed.
Little attention has been paid to lung damage from oxidative stress with respect to oxygen administration. It is clear that free radicals play an important role in the development of COPD, because 90% of all patients are exsmokers and cigarette smoke is a rich source of oxidants that cause all sorts of lung damage (234). The concentrations of oxygen administered to COPD patients are potentially toxic and may also result in lung injury caused by oxidative stress (235, 236). More research needs to be done to establish whether oxygen administration is beneficial or may contribute to lung or even peripheral tissue damage. In the meantime, if oxygen supplementation is necessary, it is recommended that the lowest effective concentration of oxygen be used (236).
CONCLUSIONS
This review underscores the fact that reduced skeletal muscle performance contributes markedly to exercise intolerance in COPD and CHF patients. Morphologic and metabolic abnormalities occur in the skeletal muscles of these patients which, in both disorders, are probably determined by the same set of contributing factors, including hypoxia, oxidative stress, disuse, medication, nutritional depletion, and systemic inflammation. Both diseases also share striking differences between peripheral muscles and the diaphragm, which may therefore require different therapeutic approaches. Future investigations of the mechanisms and relative contributions of each of the factors leading to these intrinsic muscular alterations are required.
REFERENCES
【摘要】 目的 回顾性分析肺动脉栓塞的CT表现，探讨多层螺旋CT动态增强扫描对肺动脉栓塞的诊断价值。方法 搜集经多层螺旋CT动态增强扫描确诊的肺动脉栓塞24例，回顾性分析其CT表现，重点分析肺动脉栓塞指数、中央肺动脉受累比例、有无支气管扩张显示、是否合并肺梗死。结果 24例肺动脉栓塞患者，既往有下肢深静脉血栓形成史12例，慢性心脑疾病史6例，手术、外伤或长期卧床史2例。中央肺动脉受累16例，占66.7％（16/24），支气管动脉扩张4例，占16.7％（4/24），发生肺梗死9例，占37.5％（9/24）。9例发生肺梗死的肺动脉栓塞指数24％～83％（平均46.5％），15例非肺梗死的肺动脉栓塞指数5％～61％（平均21％），两组栓塞指数具有显著差异性。结论 多层螺旋CT动态增强扫描能清楚的显示肺动脉栓塞的程度及范围，对评价病情、指导治疗和估计预后具有重要的临床价值。
【关键词】 栓塞；肺动脉；体层摄影术，X线计算机
Diagnostic value of multi-slice spiral CT dynamic contrast-enhanced scanning on pulmonary embolism
MU Qing-jin.CT Department，The First People’s Hospital of Qujing，Yunnan 655000，China
［Abstract］ Objective To evaluate the value of spiral CT dynamic contrast-enhancement scanning on pulmonary embolism by analysing the CT appearance of pulmonary embolism restrospectively.Methods Collect and analyze the appearance in multi-slice spiral CT of 24 patients with pulmonary embolism，especially the embolismic index，the involved proportion of central pulmonary arteries，the presenting proportion of bronchodilation and pulmonary infarction.Results In the 24 patients pulmonary embolism，there were 12 cases complicated with phlebothrombosis of lower limbs and 6 cases suffered from chronic cardiac and cerebral diseases，2 patients with long-term-bed-lay because of operation or trauma.In this group，16 cases were involved in central pulmonary arteries (66.7%) and 4 cases with dilated bronchial arteries (17%)，9 cases with pulmonary infraction (37.5%) whose pulmonary embolismic index was ranged from 24% to 83%， other 15 cases with no pulmonary infraction whose embolismic index was ranged from 5% to 61%，and there were significant difference between the two groups.Conclusion Multi-slice spiral CT dynamic contrast-enhanced scanning can well show the image appearance and it is an effective method for pulmonary embolism to evaluate the prognosis and direct the treatment methods.
［Key words］ embolism;pulmonary artery;tomography，X-ray computed
肺动脉栓塞是由于肺动脉或其某一分支被栓子栓塞而引起肺循环障碍的临床和病理生理综合征，是许多疾病的一种严重并发症。造成肺动脉栓塞的栓子中99％为血栓性。临床上最常见的血栓是来自下肢静脉及盆腔静脉。栓塞后如肺组织发生严重的血供障碍，可发生坏死，则称为肺梗死［1］。文献报道其发生率为10％～60％［2］。虽然现在先进诊断技术高度发展，但肺栓塞的误诊率仍在70％以上，严重影响患者的治疗和预后。未经治疗的肺动脉栓塞患者，病死率高达25％～30％，故提高对本病的认识具有重要的临床意义。笔者通过回顾性分析肺动脉栓塞患者的CT动态增强征象，旨在探讨多层螺旋CT动态增强扫描对肺动脉栓塞的诊断价值。
1 资料与方法
1.1 一般资料 笔者收集2003年1月～2006年6月临床疑有肺动脉栓塞并经多层螺旋CT动态增强扫描确诊为肺动脉栓塞的24例患者作为研究对象。男16例，女8例，年龄21～73岁，平均51岁。有突发呼吸困难或慢性呼吸困难突发加重者11例，咯血6例，发热4例，晕厥1例。其中既往有下肢深静脉血栓形成史者有12例。慢性心脑血管疾病史者6例。手术、外伤或长期卧床病史者2例。结缔组织病、肿瘤、子宫动脉栓塞术、下肢静脉滤器置入术各1例。
1.2 检查方法 设备为美国GE公司Mx8000型多层螺旋CT扫描仪及SGI后处理工作站MXVIEW软件。常规行胸部平扫加双期动态增强扫描。自胸廓入口至肋膈角下方2 cm。
扫描参数，（1）常规扫描：电压120 kV，电流80 mA，旋转时间0.75 s，螺距1.25，扫描层厚6.5 mm，重建层厚5 mm，标准重建。（2）增强扫描：电压120 kV，电流100 mA，旋转时间0.5 s，螺距1.5，扫描层厚3.2 mm，重建层厚1.6 mm，标准重建。增强扫描所用对比剂为碘海醇（300 mgI/ml），经高压注射器注射，剂量100 ml，注射流率3 ml/s，注射造影剂后分别延时27 s、60 s，行双期扫描，所得图像部分行4D重建处理。
1.3 图像分析 CT图像分析由2名高年资胸部影像诊断医师在不知道每位患者的临床资料的情况下，独立采用盲法完成。最后结果以这2名医师意见一致为准。CT征象分析内容包括：肺动脉栓塞指数、中央肺动脉受累比例、有无支气管动脉扩张显示、是否合并肺梗死等。
1.4 栓塞指数的计算 采用国外学者推荐的方法［3，4］：（1）肺动脉分为中央肺动脉（主肺动脉、双肺动脉干、双上叶动脉、双叶间动脉、双下叶动脉和中叶动脉、舌叶动脉）和外周动脉20段。（2）每一肺段动脉有充盈缺损则计1分，中央肺动脉内出现栓子则积分等于其所辖肺段动脉数（例如：左或右肺动脉计10分，叶间动脉计7分，下叶动脉计5分，上叶动脉计3分，中叶或舌叶动脉计2分）。（3）中央肺动脉受累后，其所辖远端栓塞肺动脉则不需计分。（4）设定加权系数反映血管栓塞程度。部分栓塞为1，完全栓塞为2。因此每一位患者栓塞计分最大值为40。（5）用累及计分除以40所得百分比（0～100％）作为栓塞指数反映患者栓塞的严重程度。
2 结果
本组病例中，既往有下肢深静脉血栓形成史12例，慢性心脑血管病史6例，手术、外伤或长期卧床病史2例，分别占50％（12/24）、25％（6/24）、8％（2/24）。中央肺动脉受累16例，占66.7％（16/24），周围肺动脉受累8例，占33.3％（8/12）。出现支气管动脉扩张4例，占16.7％（4/24）。出现肺动脉梗死9例，占37.5％（9/24）。9例发生肺梗死的肺动脉栓塞患者的栓塞指数24％～83％（平均46.5％）；15例非肺梗死的肺动脉栓塞患者的栓塞指数5％～61％（平均21％）。两组栓塞指数具有显著差异性。
3 讨论
3.1 肺动脉栓塞的CT表现 肺动脉内栓子的直接显示是诊断肺动脉栓塞最可靠的直接征象。在CT动态增强图像上，血栓呈低密度，与强化后肺动脉内的血液相比呈明显的密度差异。由于肺血栓本身的大小、形态不一，病程长短各异，其CT表现亦各不相同［5］。（1）部分性血栓栓塞：即血栓部分阻塞肺动脉，位于血管中央的栓子在横断面轴位CT图像上呈圆形的低密度影，周围环绕以含造影剂的高密度血流带。如与扫描层面平行，中央的栓子则呈条状低密度带，两边的造影剂则呈与之平行的高密度影，有学者称此为双轨征。本组有13例。（2）完全性血栓栓塞：即血栓基本上完全阻塞肺动脉，表现为血管腔几乎完全为低密度影占据（图1～3）。周边无环状或高密度的血流带。本组有8例。（3）环状附壁血栓型：低密度的血栓呈环形黏附于肺动脉壁，中央为强化的高密度血流。本组有3例。
3.2 肺动脉栓塞合并肺梗死的CT诊断标准 参照有关文献［6，7］：（1）初诊CT示肺内基底靠近胸膜、尖端指向肺门的楔形实变影，边界清楚，有时病灶中央见坏死组织融解后形成的含液、气空洞或呈丝瓜瓣样改变。相邻胸膜反应增厚，此为肺梗死的典型征象。（2）初诊时CT显示肺动脉栓塞患者合并肺外带实变，边界模糊，接受溶栓或抗凝治疗后至少间隔30天以上，CT复查显示病变未能完全吸收，但体积缩小，边界变清楚，形成斑片状或条索状阴影或出现肺梗死的典型征象（图4～7）。
3.3 栓塞指数与中央肺动脉受累比例的关系 肺动脉栓塞患者的病理生理改变与栓塞面积大小和栓塞程度直接相关。尽管文献报道肺血管床阻塞面积大小（栓塞指数）与肺动脉栓塞的临床严重程度不成正比［8］。但笔者认为其仍然是影响患者一系列病理生理改变的决定因素。栓塞指数越高，中央动脉受累比例越大，肺动脉栓塞患者发生严重血流动力学改变的几率越高。
3.4 栓塞指数与发生肺梗死的关系 发生肺梗死的主要原因是肺动脉栓塞，如同其他动脉一样，单一血管横断面积有80％以上被填塞，则会造成该支动脉远端血流明显减少，因此栓塞指数越大，发生肺梗死的几率就越高。本组病例中，有9例肺动脉栓塞并发肺梗死。栓塞指数为24％～83％（平均46.5％）。明显高于非并发肺梗死组的栓塞指数5％～61％（平均21％）。本组24例肺动脉栓塞病例中，有9例出现肺梗死，发生率为37.5％。肺动脉栓塞患者较少出现肺梗死，其原因主要是因为肺组织具有肺动脉系、支气管动脉系双重循环血供。
3.5 多层螺旋CT动态增强扫描对肺梗死的诊断价值 多层螺旋CT动态增强扫描能清楚的显示肺动脉的栓塞的程度几范围，是一种较可靠的检查方法。对评价病情、指导治疗和估计预后有重要的临床价值。
【参考文献】
1 马爱群.内科学.北京：人民卫生出版社，2001，71-73.
2 袁涛，于铁链，吴琦，等.肺动脉栓塞的螺旋CT肺动脉造影征象、分型及演变.临床放射学杂志，2004，23：762-765.
3 Collomb D，Paramelle PJ，Calaque D，et al.Severity assessment of acute Pulmonary temolism：evaluation using helical CT.Eur Radiol，2003，13：1508-1514.
4 Remy JM，Louvegny S，Remy J，et al.Acute central thromboembolic disease：posttherapeutic follew-up with spiral CT angiography.Rodiology，1997，203：173-180.
5 周康荣.胸部颈面部CT.上海：上海医科大学出版社，1997，122-124.
6 周旭辉，李子平，谭国胜，等.急性大面积肺动脉血栓栓塞症溶栓治疗的动态CT观察.中华放射学杂志，2005，39：256-261.
7 周旭辉，李菁，李子平，等.肺动脉栓塞中发生肺梗死的CT表现及相关因素分析.中华放射学杂志，2006，40：502-506.
8 Reid JH，Murchison JT.Acute right ventricular dilatation：a new helical CT sign of massrive pulmonary embolism.Clin Radiol，1998，53：694-698.
作者单位：655000 云南曲靖，曲靖市第一人民医院放射科
