1Program in Nutritional Metabolism and Neuroendocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston 02114; and 2General Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Submitted 18 July 2003 ; accepted in final form 9 October 2003
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ABSTRACT |
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growth hormone-releasing hormone; human immunodeficiency virus
HIV-lipodystrophy is a recently described metabolic syndrome characterized by changes in fat distribution and insulin resistance (5, 13, 14). Fat distribution changes are heterogenous and can include reduced subcutaneous fat as well as increased visceral fat. We have previously shown decreased GH secretion in patients with HIV-lipodystrophy (26), but the mechanisms of altered GH secretion in this population remain unknown. In this study, we sought to further characterize the mechanisms of reduced GH secretion among HIV-infected patients with lipodystrophy compared with age- and body mass index (BMI)-similar control groups. We investigated GH pulse dynamics and specific mechanisms related to ghrelin, somatostatin, and GHRH. Our data suggest that increased somatostatin tone, impaired GHRH stimulation of GH by excess free fatty acids (FFA), and reduced ghrelin may contribute to the altered pattern of GH secretion in HIV-lipodystrophy.
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MATERIALS AND METHODS |
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Written, informed consent was obtained from each subject before testing, in accordance with the Committee on the Use of Humans as Experimental Subjects of the Massachusetts Institute of Technology and the Subcommittee on Human Studies at the Massachusetts General Hospital. Each subject returned for a series of two outpatient and two inpatient visits to the General Clinical Research Center (GCRC) at Massachusetts General Hospital (Fig. 1). Partial data were obtained in three patients in whom not all testing was completed.
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Screening outpatient visit. After a 12-h overnight fast, subjects reported to the GCRC for a screening visit, at which time eligibility was determined on the basis of a fasting blood glucose level <126 mg/dl and a 2-h glucose level <200 mg/dl on 75-g oral glucose tolerance test.
GH assessment. Subjects returned at 0700 to the GCRC on each of 2 days separated by 1 wk for testing with either GHRH alone or a combined GHRH-arginine stimulation test (Fig. 1). The order of testing was randomized between visits; e.g., subjects were randomized to receive GHRH alone on the first visit and then combined GHRH and arginine on the second visit or vice versa. GHRH stimulation testing [GHRH-129 (Geref; Serono, Norwell, MA), 1 µg/kg iv bolus] was performed according to protocol, with GH levels collected at 15, 0, 15, 30, 45, 60, 90, and 120 min after the bolus of GHRH was given. GHRH-arginine stimulation testing was performed according to standardized protocol [GHRH-129 (Geref; Serono), 1 µg/kg iv bolus along with simultaneous administration of 0.5 g/kg arginine hydrochloride (maximum dose 30 g) iv over 30 min]. GH levels were collected at 15, 0, 15, 30, 45, 60, 90, and 120 min after GHRH administration.
Subjects returned for the fourth and fifth visits (inpatient) separated from each other by 1 wk. For each visit, subjects reported to the GCRC at 1700 for an overnight fast, with sampling performed every 20 min beginning at 1900 and ending at 0740 the next morning. Subjects were not permitted to eat after 1800 on the evening before and during frequent sampling. Subjects were randomized to receive either placebo or acipimox tablets at each visit [two 250-mg tablets of acipimox or placebo (total 500 mg) at 0200 and again at 0600]. Subjects who initially were randomized to receive acipimox for the fourth visit received placebo tablets for the fifth visit and vice versa per randomization. At 0800 (time = 0 min) on each visit, after overnight frequent sampling, a standard GHRH stimulation test [GHRH-129 (Geref; Serono), 1 µg/kg iv bolus] was performed, and GH levels were collected at 30, 60, 90, and 120 min after GHRH administration (Fig. 1). Acipimox was well tolerated by all subjects. One subject developed moderate flushing 45 min after receiving acipimox, but this resolved spontaneously 2 h later.
Body composition analysis. Height, weight, and BMI were determined in the fasting state during the first outpatient visit. Crosssectional abdominal computed tomography scanning was performed to assess the distribution of subcutaneous (SAT) and visceral abdominal fat (VAT). A lateral scout image was obtained to identify the level of the L4 pedicle, which served as a landmark for the single-slice image. Scan parameters for each image were standardized (144 cm table height, 80 kV, 70 mA, 2 s, 1-cm slice thickness). Fat attenuation coefficients were at 50 HU as described by Borkan et al. (4). VAT and SAT were then determined (4). Extremity fat was determined by dual-energy X-ray absorptiometry (Hologic 4500; Hologic, Waltham, MA).
Biochemical and immunological function. Fasting glucose, insulin, ghrelin, FFA, triglyceride, CD4 cell count, and viral load were determined on the morning of the first outpatient visit before any stimulation testing.
Laboratory methods. GH was measured by two-site radioimmunometric assay with an intra-assay coefficient of variation (CV) of 4.4% (Corning; Nichols Institute Diagnostics, San Juan Capistrano, CA). The interassay CV was 6.6%. The sensitivity of the assay was determined to be 0.05 ng/ml. IGF-I was measured by two-site radio-immunometric assay with an intra-assay CV of 4.9% (Diagnostics Systems Laboratory, Webster, TX). The interassay CV was 5.1%. The sensitivity of the assay was determined to be 2.6 ng/ml.
Ghrelin was measured by RIA (Phoenix Pharmaceuticals, Belmont, CA). The RIA uses 125I-labeled bioactive ghrelin as a tracer and a polyclonal antibody raised in rabbits against full-length, octonoylated human ghrelin as a detection probe that recognizes both the octanoyl and desoctanoyl forms of the hormone. Ghrelin levels reported in this study were assayed from serum samples. The lower limit of detection for this assay in our laboratory was 80 pg/ml. In our laboratory, the intra-assay CV was 8.7% and the interassay CV was <10%. This assay measures total ghrelin and does not specifically assess bioactive ghrelin concentration.
Nonesterified fatty acid concentrations were measured by using an in vitro enzymatic colorimetric assay kit (Wako Chemicals, Richmond, VA). The intra-assay CV for fatty acids ranged from 1.1 to 2.7%. The published normal range for fatty acids is 0.10.6 mmol/l. Insulin concentrations were measured in serum by RIA (Diagnostic Products, Los Angeles, CA). The intra- and interassay CVs range from 4.7 to 7.7 and 5.5 to 9.2%, respectively. Glucose and triglyceride concentrations were measured by standard techniques.
The CD4 count was determined by flow cytometry (Becton Dickinson Immunocytochemistry Systems, San Jose, CA), and the HIV viral load was determined by ultrasensitive assay (Amplicor HIV-1 Monitor Assay; Roche Molecular Systems, Indianapolis, IN), with limits of detection of 5075,000 copies/ml.
Pulse and deconvolution analysis: pulse and cluster programs. To assess GH pulsatility, we used Cluster, a largely model-free computerized pulse analysis algorithm to identify statistically significant pulses in relation to dose-dependent measurement error in each hormone time series (33). In performing the analysis, we specified individual test cluster sizes for the nadir and peak width of 2 (2 x 2), a minimum and maximum intraseries CV, a t-statistic to identify significant increase, and a t-statistic to define a significant decrease (32). A CV of 4.4%, the intra-assay CV for our GH assay, was used in the settings of the program. Information about the secretion of the hormone into the serum and the elimination of the hormone from the serum was obtained from PULSE 2 and PULSE 4 deconvolution and pulse detection algorithms.
Statistical analysis. Demographic and pulse characteristics among the three groups were compared by ANOVA. When the P value for the overall comparison among the three groups was 0.05, t-tests were performed comparing data among individual groups (HIV-lipodystrophic vs. HIV-nonlipodystrophic vs. normal controls). Peak GH and GH area under the curve (AUC) responses to GHRH and to combined GHRH and arginine were compared among the three groups by the nonparametric Wilcoxon test. The percent increase in peak GH response [(peak GHGHRH + arginine) (peak GHGHRH)]/ (peak GHGHRH) and percent increase in AUC GH responses between the two GH stimulation tests were compared by the Wilcoxon test. GH responses to GHRH were compared with the change in FFA before and after acipimox dosing. Fasting morning ghrelin was compared between the groups by the Wilcoxon test and with body composition, metabolic indexes, and GH pulsatility in univariate regression analysis. All statistical analyses were made using SAS JMP Statistical Database Software (version 4; SAS Institute, Cary, NC). Statistical significance was defined as a two-tailed
-value of P
0.05. Results are means ± SE unless otherwise indicated.
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RESULTS |
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GH pulse characteristics and IGF-I. Pulse dynamics were determined from frequent sampling. No differences were observed in the number of GH peaks between the groups (4.1 ± 0.6, 4.7 ± 0.8, and 4.5 ± 0.3 pulses/12 h in HIV-lipodystrophic, HIV-nonlipodystrophic, and healthy control groups, respectively; Table 2). The mean GH secretion pulse area was significantly different among the three groups (P = 0.03) and was significantly lower among the HIV-infected patients with lipodystrophy compared with the HIV-nonlipodystrophic and healthy control groups (1.14 ± 0.27 vs. 4.67 ± 1.24 ng·ml1·min, P < 0.05, HIV-lipodystrophic vs. HIV-nonlipodystrophic; 1.14 ± 0.27 vs. 3.18 ± 0.92 ng·ml1·min, P < 0.05, HIV-lipodystrophic vs. control; Table 2). The mean peak width and height were also significantly reduced in the HIV-lipodystrophic patients compared with the other groups (Table 2). IGF-I levels were 26% lower in the lipodystrophic group compared with the control group (P = 0.08) and 31% lower in the lipodystrophic group compared with the nonlipodystrophic group (P = 0.05).
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Subtraction algorithm to determine somatostatin tone. The peak GH response to GHRH was significantly lower in patients with HIV-lipodystrophy compared with HIV-nonlipodystrophic patients (2.5 ± 0.5 vs. 12.8 ± 4.6 mg/ml, P < 0.05) and compared with control patients (2.5 ± 0.5 vs. 11.3 ± 3.8 mg/ml, P < 0.05; Table 3). The AUC GH response to GHRH was also significantly reduced in the lipodystrophy group compared with the two control groups (Table 3). Percent increase in peak GH response [(peak GHGHRH + arginine) (peak GHGHRH)]/(peak GHGHRH) and percent increase in AUC GH responses between the two GH stimulation tests were significantly greater in the HIV-lipodystrophy group (Table 3).
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Responses to acipimox. Fasting morning FFA were significantly increased in the HIV-lipodystrophy group compared with HIV-nonlipodystrophic and control subjects before acipimox. The fasting FFA level decreased significantly and equivalently among all three groups in response to acipimox (Fig. 2). The change in peak GH response to GHRH before and after acipimox was significantly correlated with the change in FFA in the HIV-lipodystrophic group (r = 0.61, P = 0.04), such that response to GHRH increased most in association with the largest decrease in FFA. In contrast, the changes in FFA in response to acipimox were not associated with the changes in GH responsiveness to GHRH before and after acipimox in either the HIV-nonlipodystrophy group or in the healthy control group (Table 4).
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Ghrelin and correlation with GH pulse dynamics. The fasting ghrelin level was significantly lower among the HIV-lipodystrophy group compared with the HIV-nonlipodystrophic and control groups (Fig. 3). Fasting morning ghrelin level also correlated highly with GH mean secretion (r = 0.73, P = 0.01) and GH pulse area (r = 0.59 and P = 0.05) in the HIV-lipodystrophic but not in the other groups (Table 5). Fasting morning ghrelin correlated significantly with number of GH peaks among the HIV-infected subjects (r = 0.54, P = 0.01) and even more strongly among the HIV-lipodystrophy group (r = 0.76, P < 0.01). In contrast, no association was seen between ghrelin and GH pulsatility in the normal control group (Table 5). There was a strong negative correlation between ghrelin and fasting insulin (r = 0.57, P < 0.01) and between ghrelin and insulin AUC (r = 0.51, P = 0.02) in the HIV-infected individuals.
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DISCUSSION |
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Reduced GH secretion has been demonstrated in association with excess visceral adiposity in non-HIV-infected patients (6), and we (26) recently demonstrated that increased visceral adiposity predicted low GH concentrations in multivariate regression modeling controlling for indexes of obesity, subcutaneous fat, and overall adiposity in patients with HIV-lipodystrophy. The use of both an HIV-nonlipodystrophic group matched in terms of antiretroviral therapies and a control group similar in weight allowed us to control for the effects of HIV and antiretroviral therapy per se and focus on mechanisms of GH regulation related to fat redistribution and metabolic dysregulation. We did not study HIV-infected patients with severe primary lipoatrophy, and different results might be seen in that patient population. In contrast, we investigated patients with extreme visceral adiposity but relatively normal weight compared with previous studies of GH regulation in severe generalized obesity (11). To our knowledge, prior studies have not been performed to assess simultaneously the role of FFA, somatostatin, and ghrelin in such patients.
Our data demonstrate similar GH pulsatility but reduced GH secretion, pulse width, and area in the HIV-lipodystrophic patients compared with the other control groups. In a prior study, we investigated GH pulsatility but did not assess secretion rates by deconvolution analysis or the mechanisms of reduced GH (26). In this study, we simultaneously assessed GH pulse dynamics and performed novel studies of ghrelin, somatostatin, and GH responsivity to GHRH to assess the mechanisms of altered GH secretion in this population. GH response to GHRH was significantly blunted among the lipodystrophic patients. Furthermore, somatostatin tone was increased on the basis of the results of a subtraction algorithm, comparing the percent change in peak GH response to combined GHRH plus arginine and GHRH alone. FFA were increased in the lipodystrophic group, and GH response to GHRH was increased in proportion to the decrease in FFA by acipimox, suggesting that increased FFA inhibits GH response to GHRH in the lipodystrophic patients (Fig. 4). We also demonstrated that ghrelin is reduced in the HIV-lipodystrophic group compared with control subjects.
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Changes in fat redistribution might occur due to HIV disease or exposure to antiretroviral medications. Data from our study suggest that metabolic dysregulation in this setting, e.g., insulin resistance and increased FFA, may contribute to decreased GH through a number of different mechanisms, e.g., somatostin tone, reduced GH response to GHRH, and decreased ghrelin. In turn, decreased GH resulting from any of the mechanisms elucidated in this study may contribute to visceral adiposity and insulin resistance (18), creating a vicious cycle of metabolic dysregulation.
In humans, the neuroendocrine control of pulsatile GH secretion is poorly understood. A number of factors can modulate the pulsatile secretion of GH. These factors may influence the synthesis and secretion of GH either directly via stimulation or inhibition of pituitary somatotrophs or indirectly via GHRH and somatostatin release from the hypothalamus or by endogenous growth hormone-releasing peptides that affect the hypothalamus and/or pituitary (29). Ghrelin, a gut peptide, was recently discovered to be a natural GH secretagogue regulated by food intake (19).
Pulsatile release of GHRH is responsible for the pulsatile pattern of GH secretion observed in humans (31, 34). The frequency and amplitude of pulsatile GHRH release affect not only the pulsatile pattern of GH release but also the overall GH concentration (17). In contrast, somatostatin, a peptide produced primarily in the periventricular and medial preoptic areas of the hypothalamus, tonically inhibits GH release from the pituitary somatotrophs (1, 9).
In obese men, the mean 24-h serum GH concentration and GH pulse frequency are reduced, and daytime GH interpulse interval is lengthened compared with nonobese age-matched controls. In contrast, GH pulse amplitude is not significantly different between obese and nonobese groups (31). Furthermore, obese subjects have a significantly blunted GH response to the GH secretagogues GHRH and arginine, alone or in combination, compared with nonobese controls (11). Veldhuis et al. (35) demonstrated that the degree of body fatness was associated inversely with indexes of GH secretion. In contrast to a model of generalized obesity, we investigated patients with fat redistribution and excess visceral fat but reduced extremity fat.
Somatostatin inhibits GH release from the pituitary and is thought to be a potential mechanism for predicting reduced GH secretion in generalized obesity (11). Increased somatostatin tone might contribute to reduced GH concentration area, reduced GH pulse amplitude, and, ultimately, reduced mean GH concentrations with preserved GH pulse frequency in patients with HIV-lipodystrophy (26). To ascertain endogenous somatostatin tone, indirect techniques are needed, since currently no selective somatostatin receptor inhibitor exists for use in humans. Arginine is an amino acid that is believed to increase GH by suppression of endogenous somatostatin secretion (1, 9, 10). Investigation of somatostatin and GHRH have not, to our knowledge, been performed in subjects with excess visceral adiposity and fat redistribution.
Several groups (1, 21) have shown that arginine potentiates maximal GHRH-stimulated GH release, suggesting that arginine exerts its effects on GH secretion by decreasing somatostatin tone rather than increasing GHRH secretion. One experimental technique to determine the role of somatostatin tone in lowering GH levels in HIV-lipodystrophy is to compare the difference in GH release in response to stimulation by GHRH vs. combined GHRH-arginine (3, 9, 11). Arginine is thought to increase GH by inhibition of somatostatin tone and is therefore expected to significantly augment the stimulating response to GHRH alone when somatostatin tone is increased (9, 11). For example, arginine has been shown to augment the response to GHRH in generalized obesity through a postulated inhibition of endogenous somatostatin tone (11).
In this study, we investigated the role of somatostatin as a potential factor contributing to the observed differences in GH pulse dynamics among the lipodystrophic patients by use of a subtraction algorithm to evaluate indirectly somatostatin tone. The percent increase in stimulated GH resulting from the combined GHRH-arginine test compared with GHRH alone was largest in the lipodystrophy group, consistent with our hypothesis of increased somatostatin tone in the lipodystrophic group. Our data extend the prior data of Ghigo et al. (11), demonstrating increased somatostatin tone in the setting of severe generalized obesity, and suggest that increased somatostatin tone may also contribute to reduced GH excretion in patients with fat redistribution and visceral obesity.
A second possible mechanism to explain decreased GH secretion is altered GHRH pulsatility or GH responsivity to GHRH at the pituitary. In this regard, we hypothesized decreased GH responsivity to GHRH, as GH pulsatility was not reduced. We further hypothesized that increased FFA resulting from visceral obesity would blunt GH responses to GHRH as a potential mechanism of reduced GH secretion. Previous studies in HIV-lipodystrophic patients with visceral adiposity demonstrate increased FFA and lipolysis rates (12, 13, 15). Moreover, Hadigan et al. (15) demonstrated that increased FFA may mediate reduced insulin sensitivity in this group of patients by using acipimox to decrease FFA and acutely increase insulin sensitivity. In this study, we used acipimox as a physiological probe to acutely decrease FFA and investigate the effects of FFA manipulation on GH responsivity to GHRH. Pontiroli et al. (25) have shown in a randomized placebo-controlled trial of acipimox (500 mg) given to normal healthy subjects that GH response to GHRH was significantly greater in those who were pretreated with acipimox. Several studies have suggested that the mechanism by which the increased amounts of FFA cause diminished GH secretion is by increasing endogenous somatostatin tone (16, 20, 21). However, other studies performed in animals suggest that FFA impair GH release directly at the pituitary gland (2).
Our data demonstrate that the change in FFA in response to acipimox was highly significantly correlated with the change in peak GH responsivity to GHRH in the HIV-lipodystrophic subjects but not in the other groups. Our data suggest that excess FFA in the setting of fat redistribution and insulin resistance may impair GH responsiveness to GHRH. The tight regulation between reduction in FFA and increase in GH responsivity to GHRH in the lipodystrophic group supports this hypothesis, but further studies of the quantitative effects of higher ambient FFA concentrations in HIV-lipodystrophy are needed, for example, to determine whether there is a threshold for FFA effects on GH. Impaired GH response to GHRH would also explain the findings of normal GH pulsatility with decreased GH pulse width, pulse area, and overall secretion rate. It is also possible that increased FFA contribute to increased somatostatin tone in our subjects, as these mechanisms are not mutually exclusive. Furthermore, as suggested by Sekhar et al. (26a), increased lipolysis may occur in HIV-infected patients with more severe lipoatrophy. HIV-lipodystrophy may not be a single syndrome, and the relationship of visceral adiposity and subcutaneous fat loss is unclear. It is possible that excess FFA may alter GH secretion in the setting of severe lipoatrophy without visceral adiposity, and further studies of GH secretion in this group would be interesting. Furthermore, FFA may not be increased among all patients with HIV-lipodystrophy, and further studies of GH secretion in such patients would also be interesting. In addition, studies of HIV-infected women are necessary to investigate potential sex differences in GH regulation.
A third possible mechanism of reduced GH secretion in lipodystrophic patients is reduced ghrelin. Ghrelin is a recently discovered gut peptide that is the endogenous ligand for the GH secretagogue receptor (28). Ghrelin is nutritionally regulated and decreases with obesity (27), increases in starvation and before a meal, falls rapidly after food intake, and strongly stimulates GH secretion in humans (7). It has been demonstrated that circulating GH levels are reduced in obese subjects (30) who are insulin resistant and hyperinsulinemic (8). Recent data suggest that ghrelin is an important regulator of GH secretion during fasting as well as in response to caloric intake (24). In this study, we demonstrate reduced ghrelin in the HIV-lipodystrophy group in association with reduced mean GH pulse area, GH secretion, and increased insulin. In fact, our data suggest that insulin concentration, more than BMI, is associated with reduced ghrelin. It is possible that insulin-induced suppression of ghrelin may contribute to reduced GH secretion in HIV-lipodystrophy. However, the relationship between insulin and ghrelin is unclear in normal physiology, and it remains uncertain whether insulin per se or pattern of macronutrient ingestion affects ghrelin. Alternatively, increased somatostatin tone may contribute to low ghrelin concentration. Low ghrelin may be a marker of metabolic dysregulation and altered body fat without specifically contributing to low GH. Determination of a single fasting ghrelin level may be inadequate, and further studies are necessary to determine meal-related ghrelin responses in relationship to GH in HIV-lipodystrophy. In addition, our study was limited to the determination of total ghrelin, whereas measures of bioactive ghrelin may also be useful. Further studies will be needed to investigate the specific role of reduced ghrelin in mediating low GH in conditions of visceral adiposity.
Our data demonstrating reduced GH secretion, peak area, and preserved GH pulsatility are similar to those of Magiakou et al. (22). Although urine free cortisol levels and other tests of the hypothalamo-pituitary-adrenal axis have not been shown to be abnormal in HIV-lipodystrophy (23, 26), it is possible that patients with HIV-lipodystrophy may have glucocortical hypersensitivity at the level of the glucocorticoid receptor. Further studies are needed to investigate whether there is a relationship between altered glucocorticoid and GH dynamics in this population.
In conclusion, this study helps elucidate the physiological mechanisms of reduced GH secretion in HIV-lipodystrophy. In a group of lipodystrophic patients with visceral obesity and fat redistribution, we demonstrate an equivalent number of GH pulses but markedly reduced GH secretion, pulse area, and GH pulse width compared with control subjects. Our data suggest increased somatostatin and an effect of excess FFA to impair GH response to GHRH. Furthermore, we demonstrate low ghrelin, which may also contribute to low GH in this population. Taken together, these data suggest a complex schema whereby alterations in insulin and fatty acids may affect GH secretion in HIV-lipodystrophy.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported in part by National Institutes of Health Grants R01 DK-063639 and M01 RR-01066.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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