Reduced free IGF-I and increased IGFBP-3 proteolysis in Turner syndrome: modulation by female sex steroids

Claus Højbjerg Gravholt, Jan Frystyk, Allan Flyvbjerg, Hans Ørskov, and Jens Sandahl Christiansen

Medical Department M (Endocrinology and Diabetes) and Medical Research Laboratories, Aarhus University Hospital, DK-8000 Aarhus C, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The bioactivity of the growth hormone-insulin-like growth factor (IGF) system is reduced in Turner syndrome and may explain the reduction seen in final height. We compared levels of free and total IGF-I, immunoreactive and Western ligand blot IGF-binding protein (IGFBP)-3, and IGFBP-3 proteolysis in women with Turner syndrome (n = 23) before (TB) and during 6 mo treatment with 17beta -estradiol and norethisterone. An age-matched group of controls (n = 24) was included. Total IGF-I and immunoreactive levels of IGFBP-3 were comparable in TB and controls, whereas free IGF-I (P = 0.02) in TB was less than in controls. Western ligand blotting (WLB)-IGFBP-3 was significantly lower in TB than in controls (P = 0.0005). Accordingly, IGFBP-3 proteolysis was greater in Turner syndrome (P = 0.001). Female sex steroid treatment increased WLB-IGFBP-3 (P = 0.0005), whereas immunoreactive IGFBP-3 and IGFBP-3 proteolysis were normalized (P = 0.004). Free IGF-I remained unchanged (P = 0.8), with a tendency toward a decrease in total IGF-I (P = 0.1). In conclusion, despite normal total IGF-I and immunoreactive IGFBP-3, free serum IGF-I is less and IGFBP-3 proteolysis is greater in Turner syndrome than in controls. During sex steroid treatment, IGFBP-3 proteolysis normalized, without any change in free IGF-I.

Turner syndrome; total insulin-like growth factor I; free insulin-like growth factor I; insulin-like growth factor-binding protein 3; proteolysis; 17beta -estradiol; final height


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TURNER SYNDROME IS CHARACTERIZED primarily by reduced final height, gonadal insufficiency, and infertility. Because of the reduction in final height, interest has focused on the growth hormone (GH)-insulin-like growth factor (IGF) axis. However, results are equivocal; the spontaneous and stimulated GH secretion has been found to be diminished by some (35, 39), whereas others have found a normal GH secretion (38, 42, 50). Also, the bioactivity of the circulating GH has been reported to be reduced (16), and different GH isoforms have been found to prevail by some (7) but not by others (30). The normal pubertal increase in GH secretion is absent in Turner girls but can be restored partially by replacement of sex hormones (41, 51). IGF-I is the effector hormone of some of the growth actions of GH (11). In girls with Turner syndrome, levels of serum total (extractable) IGF-I levels have been found to be lower than (41) or comparable to age-matched controls (51). Low-dose estrogen therapy has been shown to increase IGF-I (9, 40), whereas in pharmacological doses estrogen suppresses circulating levels of IGF-I in normal individuals (49). Untreated adult patients with Turner syndrome have normal serum levels of total IGF-I and -II. Similarly, serum levels of IGF-binding protein (IGFBP)-1, -2, and -3 are within the normal range (23). However, in normal adult women, estrogen therapy has been shown to increase IGFBP-1, which is an important regulator of free IGF-I. Estrogen therapy increases IGFBP-1 (47, 48), which would tend to lower free IGF-I since IGFBP-1 is considered an inhibitory IGFBP (8), and estrogen also increases GH (46), which on the contrary would be expected to increase free IGF-I. At present, there is no information on serum levels of free IGF-I in adults with Turner syndrome, and it is unknown how free IGF-I is affected by female sex steroid therapy.

In pregnancy, a condition characterized by high levels of female sex steroids, there is a marked degradation in serum IGFBP-3 in vivo and in vitro (28). This proteolysis disappears after delivery. It is the general belief that IGFBP-3 proteolysis increases the bioavailability of IGF-I (28, 32). In Turner syndrome, characterized by extreme hypoestrogenism, IGFBP-3 proteolysis has never been studied.

The aim of the present study was to compare the immunoreactive circulating levels of IGFBP-1, -2, and -3, IGFBP-3 proteolysis, and circulating levels of free and total IGF-I and IGF-II in adults with Turner syndrome and in healthy controls. Because immunoreactive levels of IGFBP-3 may include inactive fragments of the binding protein not able to participate in the ternary complex, we chose to also study IGFBP-3, determined by Western ligand blotting (WLB), which supposedly shows intact IGFBP-3. Furthermore, the effects of sex hormone replacement therapy on the above-mentioned parameters were studied. The study was performed as part of a clinical trial investigating the effect of 17beta -estradiol and norethisterone administration on the GH-IGF axis and carbohydrate and lipid metabolism in adult Turner syndrome (23-25).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. The study consisted of 23 (34.9 ± 8.2 yr) patients with Turner syndrome and a control group of 24 (32.7 ± 7.6 yr) normal women with presumed normal karyotype. The karyotypes of the patients were 45,X (n = 15), 45,X/46,X,i(Xq) (n = 2), 46,X,i(Xq) (n = 2), 45,X/46,XY (n = 2), 45,X/46,XX (n = 1), and 46,X,del(Xq) (n = 1). Some Turner patients had previously participated in scientific studies, whereas others were recruited through the National Society of Turner Contact Groups in Denmark. The controls were recruited among female blood donors by advertisement in the blood donor clinic at Aarhus University Hospital. The control group was matched with respect to age. Exclusion criteria were former or present chronic disease, severe obesity [body mass index (BMI) >40], and heavy smoking (>20 cigarettes/day). All subjects gave informed consent, and the protocol was approved by the Aarhus County Ethical Scientific Committee and the Danish Health Authorities.

Design. All patients were receiving female hormone replacement therapy, which was discontinued 4 mo before the initial examination (basal examination of Turner patients). After the initial evaluation, patients were randomized to one of two regimens of hormone replacement for 6 mo (treatment examination of Turner patients): oral hormone substitution consisting of 2 mg 17beta -estradiol/day from days 1 to 12 of the menstrual cycle, 2 mg 17beta -estradiol/day and 1 mg norethisterone acetate/day from days 13 to 22, and 1 mg 17beta -estradiol/day from days 23 to 28 (Trisekvens; Novo Nordisk, Bagsværd, Denmark) or transdermal estrogen substitution consisting of ~50 µg 17beta -estradiol · 55 kg-1 · day-1 for 28 days (Estraderm; Ciba-Geigy) and 1 mg norethisterone (Noretisteron Dak; Nycomed DAK, Copenhagen, Denmark) administered orally from days 13 to 22. Ten subjects were randomly allocated to the group receiving transdermal estrogen, and 13 subjects were allocated to the group receiving oral estrogens. A detailed statistical analysis showed no significant differences between the two treatment groups regarding the studied parameters; consequently results in the two groups were pooled.

All patients were studied before and after 6 mo of sex hormone therapy, whereas the control group was evaluated one time. Control subjects were studied in the early follicular stage (days 5-10) of the menstrual cycle; Turner subjects were studied on days 5-10 of the hormone replacement therapy cycle while receiving estrogen only.

Methods. Bioelectrical resistance and impedance were measured, and, based on this, fat mass, fat-free mass (FFM), and total body water (TBW) were estimated according to algorithms provided by the manufacturer (Animeter; HTS-Engineering, Odense, Denmark; see Ref. 26). Body mass index was calculated as weight (kg) divided by squared height (m2), and the waist-to-hip ratio was determined in the supine position.

A cannula was inserted in a cubital vein, and fasting blood samples were drawn at 0800 after an overnight fast (10-12 h). Twenty-four-hour blood sampling was started at 1200 and was continued every 20 min. Meals were served at 1230, 1500, 1800, 2100, and 0800. Blood samples were collected during standardized conditions. All samples were analyzed for GH. The integrated concentration of GH (ICGH) was calculated using the trapezoidal rule; these data, and data on pulsatility and regularity of GH secretion, have been published previously (23, 25). All other analyses were performed in fasting samples. Serum was separated and stored at -20°C until assayed.

Assays for free and total IGF-I and -II, IGFBP-1, IGFBP-2, IGFBP-3, GH, and insulin. All samples were analyzed in duplicate in the same run. Serum total IGF-I and IGF-II were measured by in-house noncompetitive, time-resolved immunofluorometric assays after acid-ethanol extraction of serum as previously described (18).

Free IGF-I and IGF-II were separated from bound IGFs by ultrafiltration (20); serum samples were diluted 1:11 in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5% human serum albumin, and 600 µl of the dilution were applied to a YMT-30 ultrafiltration membrane mounted in an MPS-1 supporting device (both from W. R. Grace, Amicon, Beverly, MA) and centrifuged at 3,000 g at 37°C in triplicate. After appropriate dilution of the filtrate, the concentrations of free IGF-I and IGF-II were measured directly in the time-resolved immunofluorometric assays. All samples in the study were run in the same assay. The detection limit in serum was 27.5 ng/l for free IGF-I and 55 ng/l for free IGF-II. The average intra- and interassay coefficients of variation (CV) were 14 and 17%, respectively.

Serum IGFBP-1 was measured by ELISA (Medix Biochemica, Kainainen, Finland). IGFBP-2 was measured by RIA and IGFBP-3 by immunoradiometric assay (Diagnostic System Laboratories, Webster, TX).

GH was measured with a double monoclonal immunofluorometric assay (Wallac Oy, Turku, Finland). The interassay CV in samples varied between 1.7 and 2.4%, the intra-assay CV varied between 1.9 and 3.0% for GH concentrations of 12.08 and 0.27 µg/l, and the detection limit was 0.01 µg/l. The 24-h ICGH was calculated by use of the trapezoidal rule as the area under the curve.

Serum insulin was measured by ELISA employing a two-site immunoassay, which does not detect proinsulin, split-(32---33)-proinsulin, and des-(31---32)-proinsulin, whereas split-(65---66)- and des-(64---64)-proinsulin cross-react 30 and 63%, respectively. The intra-assay CV was 2.0% (n = 75) at a serum level of 200 pM.

WLB. One serum sample from each participant was subjected to WLB to attain additional confirmation of the immunoreactive IGFBP-3 level. SDS-PAGE and ligand blot analysis were performed in serum from all patients and controls according to the method of Hossenlopp et al. (29), as previously described (14). Two microliters of serum were subjected to SDS-PAGE (10% polyacrylamide) under nonreducing conditions. Specificity of the IGFBP-3 band was ensured by competitive coincubation with unlabeled recombinant human IGF-I purchased from Bachem (Budendorf, Switzerland).

125I-labeled IGFBP-3 degradation assay. The IGFBP-3 protease assay was performed as previously described (15). In short, I25I-labeled IGFBP-3 (30,000 counts/min; Diagnostic System Laboratories) was incubated for 18 h at 37°C with 2 µl of serum from patients and controls and subjected to SDS-PAGE as described above. On each gel, serum samples from a healthy nonpregnant subject and a term-pregnant woman were used as internal controls. Gels were fixed in a solution of 7% acetic acid, dried, and autoradiographed. The degree of proteolysis was calculated as a ratio of the absorbency of fragmented I25I-IGFBP-3 (30-, 20-, and 16-kDa bands) over the sum of all I25I-IGFBP-3 (38- to 42-, 30-, 20-, and 16-kDa bands)-related optical densities in that lane and expressed as a percentage.

Quantification of WLB and protease assay. Autoradiograms were quantified by densitometry using a Shimadzu CS-9001 PC dual-wavelength flying spot scanner. The relative density of the bands was measured as arbitrary absorbency units per square millimeter (AU/mm2).

Statistical analysis. All statistical calculations were done with SPSS for Windows version 7.5 (SPSS, Chicago, IL). Data were examined by Student's two-tailed unpaired and paired t-tests or the Mann-Whitney or Wilcoxon two-tailed tests when appropriate. Multiple backward stepwise linear regression was used to examine the principal determinants of IGF-I, and adjusted r values are presented. Results are expressed as means ± SD. P values <5% were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GH secretion, body composition, and age. Anthropometric data on Turner patients and controls have been presented previously (23). In Table 1, pertinent data are presented. In brief, Turner women and controls were matched for age, but not for BMI, because of the characteristic anthropometry and body composition of the adult Turner syndrome (22). Although BMI was slightly higher in Turner patients than in the control group, FFM was significantly higher in controls. ICGH was significantly decreased in untreated Turner syndrome (Table 2). BMI was unchanged by treatment with sex steroid substitution, whereas FFM and ICGH increased significantly.

                              
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Table 1.   Number and age of participants and levels of anthropometric data in Turner patients at baseline and during sex hormone replacement and in normal women


                              
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Table 2.   Levels of variables from the GH-IGF axis in Turner patients at baseline and during hormone replacement therapy and in normal women

Circulating levels of IGFs and IGFBPs: untreated Turner syndrome and controls. The level of free IGF-I was significantly lower by 25% in Turner syndrome (Table 2), despite comparable levels of total IGF-I. Like total IGF-I, free IGF-I decreased with age in both Turner patients and controls (Fig. 1). The level of free IGF-II was significantly higher in Turner patients than in controls, whereas the level of total IGF-II was similar in the two groups (Table 2). Circulating levels of immunoreactive IGFBP-1, -2, and -3 were comparable in Turner syndrome and controls. Intact IGFBP-3, as determined by WLB, was significantly reduced by 27% in Turner syndrome, indicating significant proteolysis. Mirroring these changes in vitro, IGFBP-3 proteolysis was almost twofold higher in serum from Turner patients (Table 2). The karyotype of the women with Turner syndrome influenced the in vitro proteolysis and was most pronounced in the group of women with karyotypes other than 45,X (45,X vs. other karyotypes: 24.0 ± 7.2 vs. 37.5 ± 13.7%, P = 0.006). IGFBP-3 determined by WLB was not affected by the karyotype of the Turner women. In Turner women, free IGF-I correlated significantly and positively with WLB- IGFBP-3 (r = 0.752, P = 0.0005) and inversely with IGFBP-3 proteolysis (r = -0.520, P = 0.013; Fig. 2, A and C), whereas free IGF-II correlated negatively with WLB-IGFBP-3 (r = -0.617, P = 0.002; Fig. 2B). In controls, there were no significant correlations between free IGFs and any of the IGFBPs. In both Turner and control women, free IGF-I correlated significantly with total IGF-I (total IGF: r = 0.605, P = 0.002; controls: r = 0.767, P = 0.0005). There were no significant correlations between free IGF-II and total IGF-II.


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Fig. 1.   Free insulin-like growth factor (IGF)-I (A) and total IGF-I (B) in untreated patients with Turner syndrome () and normal women (open circle ) vs. age. Solid line, Turner syndrome; dashed line, normal women.



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Fig. 2.   Free IGF-I (A) and free IGF-II (B) in patients with untreated Turner syndrome () and normal women (open circle ) vs. Western ligand blotting (WLB) and IGF-binding protein (IGFBP)-3 and free IGF-I vs. IGFBP-3 (C). Solid line, Turner syndrome; dashed line, normal women.

Circulating levels of IGFs and IGFBPs: untreated vs. treated Turner syndrome. Free IGF-I was unchanged by treatment with sex steroid replacement therapy, whereas there was a tendency toward a decrease in total IGF-I (P = 0.1), total IGF-II (P = 0.06), and IGFBP-3 (P = 0.06; Table 2). Free IGF-II decreased significantly (P = 0.02). IGFBP-1 increased significantly, whereas IGFBP-2 and -3 and insulin were unchanged. There was a significant increase in IGFBP-3 as ascertained by WLB (Table 2). Concomitantly, in vitro IGFBP-3 proteolysis normalized (Table 2). In the treated situation, there were no significant correlations between free IGF-I and any of the IGFBPs, whereas free IGF-II was associated positively with IGFBP-3 proteolysis (r = 0.493, P = 0.02). Free IGF-I correlated significantly with total IGF-I (r = 0.756, P = 0.0005).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Increased IGFBP-3 proteolysis and, conversely, a decreased level of IGFBP-3 as determined by WLB, mirroring the increased proteolysis, are reported for the first time in patients with Turner syndrome. IGFBP-3 proteolytic activity has been reported in a number of conditions. It was first reported in pregnancy (28) and later in severe illness (13), after surgery (10, 12), and in type 2 diabetes (2). Furthermore, insulin resistance has been suggested to play a part in IGFBP-3 proteolysis (4). Plasminogen may act to induce IGFBP-3 proteolysis in nonpregnancy serum, whereas it is not clear what causes the enhanced proteolysis in pregnancy serum (3). In postmenopausal women, who also have very low levels of circulating levels of sex hormones, there is no evidence for increased IGFBP proteolysis (27). In addition, the mechanisms by which substitution with 17beta -estradiol and norethisterone virtually abolish the additional IGF-BP-3 proteolysis observed in Turner syndrome are not clear. One explanation may be that sex hormone treatment normalizes some intrinsic hepatic malfunction; several other hepatic enzyme systems, such as alkaline phosphatase, gamma -glutamyl transferase, and alanine aminotransferase, are increased in untreated Turner syndrome (23, 45), and they are normalized during treatment with sex hormones (23). It has also been suggested that IGFBP-3 proteolysis may increase the bioavailability of IGF-I in the circulation (28, 32); however, this is not supported by the present data, since free IGF-I was inversely correlated to the degree of IGFBP-3 proteolysis. This inverse correlation may, however, be explained by the fact that free IGF-I has a very short half-life (~14 min) compared with binary complex-bound IGF-I and IGF-I circulating in the ternary complex (19), by increased binding to IGF-I receptors, and by increased clearance of IGF-I; i.e., when more IGF-I is released from the ternary or binary complex, lower levels are the resultant effect of the very short half-life of free IGF-I. We speculate that shorter-term changes in IGFBP-3 proteolysis, i.e., pregnancy, severe illness, and surgery, may increase the bioavailability of IGF-I, whereas more chronic conditions, type 2 diabetes, insulin resistance, and the severe hypoestrogenism of Turner syndrome, may decrease the bioavailability of IGF-I. Free IGF-I is thought to be the bioavailable or active part in serum of the total measurable IGF-I. So far, circumstantial evidence supports this contention, since obese patients have suppressed GH levels, normal total IGF-I, but high free IGF-I, which could explain the normal growth in obese children despite suppressed GH (21). Furthermore, GH treatment of GH-deficient patients increases free IGF-I in conjunction with total IGF-I (44), and finally, bioactive somatomedin C (IGF-I) shows the same diurnal variation as free IGF-I (37). In the present study, we found women with Turner syndrome to have reduced levels of free IGF-I, normal total IGF-I, and low diurnal GH levels despite a higher BMI and a lower FFM compared with an age-matched control group. This means that Turner women are different from moderately obese women who also have low GH levels, normal total IGF-I, but high free IGF-I (21). Recent evidence shows that IGF-II has a role as a permissive factor for IGFBP-4 proteolysis and subsequent "release" of (bioactive) IGF-I from IGFBP-4, at least in osteoblasts (33). If IGF-II also works as a permissive cofactor in the proteolysis of other IGFBPs in the circulation, this could explain the high levels of free IGF-II in Turner syndrome. Furthermore, the highly significant decrease in free IGF-II during sex hormone treatment was seen in parallel with a highly significant increase in WLB-IGFBP-3 and a decrease in the proteolysis of IGFBP-3.

It is unknown why Turner patients are growth retarded. In girls with Turner syndrome, circulating androgens are reduced (1). Recently, in a murine model, it has been shown that the effect of testosterone on condylar growth is partly mediated by IGF-I (34). Furthermore, a very recent clinical study of Turner girls has shown that treatment with very high doses of GH can indeed normalize the final height in Turner syndrome (43). In this context, it is of interest that fragments of IGFBP-3 can inhibit growth of cells, an effect that is independent of IGF-I and the IGF-I receptor (5). By combining these findings with the present results, low free IGF-I and high circulating levels of fragments of IGFBP-3 provide a picture of a partially defunctioning GH-IGF-IGFBP axis. As shown here, another axis, the sex hormone axis, seems to be able to modulate the GH-IGF-IGFBP axis, making the picture very intriguing indeed. Therefore, it may be speculated that the reduction in circulating free IGF-I and the greater IGFBP-3 proteolysis found in the present study are part of the explanation for growth failure in Turner syndrome. If these alterations are also present in the microenvironment in all tissues where growth should occur and thus the autocrine and paracrine effects of the hormone are similarly reduced, the growth potential of these tissues, i.e., bone, cartilage, and connective tissue, may be reduced, and the resultant reduction in growth could potentially explain part of the perturbed height in Turner syndrome.

Oral ethynyl estradiol, conjugated estrogen, or 17beta -estradiol has previously been found to be associated with a decrease in total IGF-I in postmenopausal women (27, 46), whereas transdermal 17beta -estradiol has been associated with either an increase (46), no effect (6, 27), or a decrease in total IGF-I (17). In the present study, there was a tendency toward a decrease in total IGF-I in both the orally and the transdermally treated groups, whereas there was no effect of treatment on free IGF-I. To our knowledge, free IGF-I has not previously been investigated during female sex hormone replacement in postmenopausal women or in other hypogonadal groups. Thus it appears that free IGF-I is not influenced by female sex hormones, and one can speculate that the decrease in total IGF-I seen in trials involving postmenopausal women is merely a phenomenon attributable to changes that do not affect the free or the putatively bioavailable fraction of IGF-I. In a clinical setting this makes sense, since we and others have found that replacement with female sex hormones is associated with favorable changes in body composition and increases in physical fitness and muscle strength (23, 31, 36), and these findings would be difficult to accept in the face of a decline in IGF-I levels. It will therefore be of considerable interest to examine the level of free IGF-I in trials of female sex hormone substitution in postmenopausal women.

In conclusion, women with Turner syndrome have decreased levels of free IGF-I and WLB-IGFBP-3 and increased levels of free IGF-II and IGFBP-3 proteolysis. Replacement with sex steroids normalizes the circulating levels of WLB-IGFBP-3 while having no effect on free IGF-I but decreasing free IGF-II. It may be speculated that the high degree of IGFBP-3 proteolysis is part of the explanation for the blunted growth response to exogenous GH treatment during puberty. Furthermore, the widely different degrees of IGFBP-3 proteolysis, as seen in the current population and if verified in a prepubertal population, may explain the highly variable growth response to GH treatment in young Turner patients.


    ACKNOWLEDGEMENTS

Karen Mathiasen, Kirsten Nyborg, Lone Korsgaard, and Eva Seier Christoffersen are thanked for expert technical help. Ciba-Geigy and Novo Nordisk are thanked for the generous gift of Estraderm and Trisekvens.


    FOOTNOTES

C. Højbjerg Gravholt was supported by a research fellowship by the University of Aarhus. The study was supported by a grant from the Danish Diabetes Association and by Danish Health Research Council Grant nos. 9600822 (Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration) and 9700592 (A. Flyvbjerg).

Address for reprint requests and other correspondence: C. Højbjerg Gravholt, Medical Dept. M (Endocrinology and Diabetes), Århus Kommunehospital, DK-8000 Aarhus C, Denmark (E-mail: ch.gravholt{at}dadlnet.dk).

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.

Received 10 July 2000; accepted in final form 4 October 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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