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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 5 1516-1521
Copyright © 1997 by The Endocrine Society

Experimental Studies

Circulating Non-22-Kilodalton Growth Hormone Isoforms in Acromegalic Men before and after Transsphenoidal Surgery1

Cesar L. Boguszewski, Gudmundur Johannsson, Bengt-Åke Bengtsson, Anders Johansson, Björn Carlsson and Lena M. S. Carlsson

Research Centre for Endocrinology and Metabolism, Department of Internal Medicine (C.L.B., G.J., B-Å.B., B.C., L.M.S.C.), and Department of Radiology (A.J.), Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden

Address all correspondence and requests for reprints to: Cesar L Boguszewski, Research Centre for Endocrinology and Metabolism, Sahlgrenska University Hospital, Bruna Stråket, 16, S-413 45 Göteborg, Sweden.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
GH represents several molecular isoforms in addition to the main 22-kDa (22K) GH. There have been reports suggesting that circulating non-22K GH isoforms are increased in acromegaly, but the possible implications of such observations in the management of the disease have not been addressed. The aim of this study was to evaluate the proportion of circulating non-22K GH isoforms in acromegaly. In addition, the relationships between the amount of non-22K GH and tumor size, biochemical measurements, and body composition also were investigated. Samples with different GH levels were selected from 24-h GH profiles from 15 acromegalic men evaluated before and 1 yr after transsphenoidal surgery and from 13 healthy men. The serum non-22K GH levels, expressed as percentage of total GH concentration, were determined by the 22K GH exclusion assay, which is based on immunomagnetic extraction of 22K GH from serum and quantitation of non-22K GH using a polyclonal GH assay. The proportion of non-22K GH isoforms was fairly constant in different samples from the same patient, regardless of the GH level. However, a wide variation of values was observed among acromegalics, both before (14–51%) and after surgery (8–62%). The proportion of non-22K GH isoforms was increased in untreated patients, compared with controls (26.6 vs. 17.4%; P < 0.01), and the values correlated significantly to tumor size, mean 24-h GH concentration, serum PRL, and extracellular water. After surgery, patients not truly cured, with mean 24-h GH concentration of 1 µg/L or more, had an increased proportion of non-22K GH, compared with those with levels less than 1 µg/L (P < 0.01). In the former group, the median values were similar than those in untreated acromegalics (34 vs. 26.6%, respectively), whereas in the latter, they were comparable with those in the controls (15.2 vs. 17.4%, respectively). We conclude that acromegalics have an increased proportion of circulating non-22K GH isoforms. The values are fairly constant in different samples from an individual, regardless of GH level, but a large spectrum can be observed among patients. This variability suggests that different pituitary adenomas secrete GH isoforms in variable amounts. Our observation that a higher proportion of non-22K GH isoforms is present in patients not truly cured after surgery suggests that the evaluation of non-22K GH isoforms can be useful in the follow-up of acromegalic patients.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
HUMAN GH consists of several molecular isoforms, in addition to the predominant 22-kDa (22K) GH, which are observed both in blood and in pituitary extracts (1, 2, 3, 4). The GH heterogeneity contributes to the discrepancies observed when plasma GH concentrations are measured by different assays (5, 6, 7). Although the physiological role of the various non-22K GH isoforms is not completely known, some of them clearly differ from 22K GH, regarding biological activity, binding properties, and metabolic clearance (8, 9, 10, 11, 12). It is established that GH induces sequential dimerization of the GH receptor (GHR). First, site 1 on GH binds to one GHR molecule and then site 2 binds to a second GHR molecule (13, 14). On the basis of this mechanism, GH isoforms and fragments lacking binding site 1 or 2 may interact as partial agonists and/or antagonists of the GHR (15, 16). In addition, it has been suggested that the effects of some non-22K GH isoforms and fragments might be mediated by specific receptors (10, 15, 17).

In acromegaly, there have been reports on the lack of correlation between GH and insulin-like growth factor 1 (IGF-1) levels (18, 19, 20), which have prompted some investigators to evaluate the proportion of GH isoforms in these patients, because disparity between bioactive and immunoreactive GH in blood could be a potential explanation for these apparent discrepancies. Using immunoadsorbent chromatography, Baumann et al. (21) found that the relative percentages of GH monomeric isoforms in serum did not differ between normal individuals and acromegalics. In contrast, studies using gel filtration, affinity chromatography, and GH immunoreactivity to different site-specific antibodies have revealed an increased proportion of circulating non-22K GH isoforms and the presence of abnormal GH or GH-like molecules in patients with acromegaly (22, 23, 24, 25).

The aim of this study was to evaluate the proportion of non-22K GH isoforms in serum samples with different GH concentrations obtained from acromegalic men before and after surgery and to compare the results with those in healthy male subjects, using the recently developed 22K GH exclusion assay (GHEA) for measurement of non-22K GH isoforms in blood (26). We also have examined the proportion of circulating non-22K GH and its relation to tumor size, body composition, and circulating factors involved in growth and metabolism.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Fifteen men, 25–75 yr old (mean, 48.5 ± 3.7 yr), with a body mass index of 27.6 ± 0.9 kg/m2, surgically treated for GH-producing adenoma between 1988–1994 at our hospital, were studied. The diagnosis of acromegaly was based upon classical clinical findings, elevated serum GH levels with typical abnormalities in the 24-h GH secretory pattern (20), and high serum IGF-1 levels. Thirteen healthy men, 44–63 yr old (52.9 ± 1.8 yr), body mass index 26.5 ± 1.1 kg/m2, were used as control group for biochemical analysis and 24-h GH profiles.

Study protocol

In the initial diagnostic work-up, laboratory examination, including a 24-h GH profile, was performed. Body weight (BW) was measured in the morning with the subjects wearing indoor clothing, and body height was measured barefoot. Body composition was assessed according to the four-compartment model. All patients were thereafter treated with transsphenoidal surgery for the GH-producing tumor. Postoperative evaluation was performed within 3 months after surgery for exclusion of pituitary hormonal deficiencies. One year after surgery, the initial evaluation was repeated, and the acromegalic patients were divided into two groups according to the mean 24-h GH concentration in the profile: 1 µg/L or more (group A) and less than 1 µg/L (group B). Table 1Go shows the laboratory measurements, 24-h GH profile, and body composition findings in the study groups. All subjects gave their informed consent, and the study protocol was approved by the Ethics Committee of the University of Göteborg.


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  		Table 1. Laboratory measurements, 24-h GH profile, and body composition in the control group, untreated acromegalics, and acromegalics evaluated 1-yr after transsphenoidal surgery who presented a 24-h GH secretion rate >=1 µg/L (group A) or <1 µg/L (group B)

 
24-h GH profiles

Blood samples for 24-h GH profiles were collected as previously described (27). The area under the curve, the maximum GH peak, and the mean 24-h GH concentration were calculated using the Pulsar program (28). The first sample in the morning was used to measure IGF-1 (IGF-1 SD scores adjusted for sex and age reference values were calculated, according to Ref. 29), IGF binding protein-1 (IGFBP-1), IGFBP-3, insulin, and PRL. In the acromegalics, non-22K GH levels were measured in three different samples: a peak sample, collected at the time of the maximum secretory GH peak; a middle sample, defined as the sample with a GH level corresponding to the mean GH concentration during the 24-h period; and a nadir sample (only presurgery), representing the sample with the lowest detectable GH level in the profile. In one patient, no peak or middle samples were available after surgery. Thus, a total of 73 samples (45 presurgery and 28 post surgery) were selected for measurements of non-22K GH. In two middle samples, non-22K GH levels were undetectable after serum extraction of 22K GH, and they were excluded from the analysis. In the control group, non-22K GH levels were determined only in peak samples because of the very low GH concentrations in middle and baseline samples.

Body composition (four-compartment model)

The body composition was estimated according to the four-compartment model (30). In this model, the BW consists of the sum of body cell mass (BCM), extracellular water (ECW), fat-free extracellular solids, and body fat (BF), assessed from total body potassium, total body water (TBW), BW, and the normal BW. Reference values were derived from regression equations from body composition studies in 134 randomly selected healthy men, 20–70 yr old. Individual observed/predicted value ratios for current BW, age, and gender (expressed as a percentage of the predicted value) were calculated for BCM (BCM%), TBW (TBW%), ECW (ECW%), and BF (BF%).

Assays

GH levels were measured using a polyclonal immunoradiometric assay (IRMA, Pharmacia & Upjohn, Uppsala, Sweden). The GH standards provided in the IRMA were calibrated against the 1st International Reference Preparation from the WHO (IRP 80/505), in which 1 mg = 2.6 IU of GH (31). IGF-1 concentrations were determined both in the patients and in the reference population using a hydrochloric acid-ethanol extraction RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA). IGFBP-3 levels were measured by RIA (Nichols Institute, Wijehen, The Netherlands). IGFBP-1 levels were determined by IRMA (Active IGFBP-1; Diagnostic Systems Laboratories, Webster, TX). Insulin levels were measured by RIA (Insulin RIA 100, Pharmacia & Upjohn). PRL concentrations were determined by RIA (Diagnostic Products Corporation, Los Angeles, CA).

22K GHEA

The GHEA, which has been previously described (26), was used to determine the serum levels of non-22K GH isoforms. The GHEA is based upon the extraction of monomeric and dimeric 22K GH from serum using an anti-22K GH monoclonal antibody (MCB; Genentech Inc., San Francisco, CA) and magnetic beads coated with rat antimouse IgG (batch number: A2300; Dynal, Oslo, Norway). After 22K GH extraction, the levels of the remaining non-22K GH isoforms are measured by a polyclonal IRMA (Pharmacia & Upjohn), calibrated against the IRP 80/505 (31). In the GHEA, total GH levels are determined in a set of samples incubated only with assay buffer (without addition of MCB), and non-22K GH isoforms are expressed as percentage of the total GH concentration in the samples. The assay has a detection limit of 0.02 µg/L. In this study, the between-assay CVs for 22K GH extraction were below 5%, as determined in GH-free serum spiked with monomeric or dimeric 22K GH, as well as in two different serum pools with known GH concentrations.

Tumor size

Twelve patients were examined by magnetic resonance imaging and three patients by computed tomography (CT). The tumor size was determined retrospectively from the preoperative images by measuring the sagital (S), coronal (C), and transverse (T) diameters and applying the formula SxCxT/2 to approximate the tumor size. In one patient with a large adenoma, the resolution of preoperative CT image did not allow the direct estimation of the tumor size. Instead, the size of this macroadenoma was estimated by comparison with the CT image of another macroadenoma. The parasellar extension of the tumor was graded from 0–4, according to the classification proposed by Knosp et al. (32).

Statistical analysis

All the descriptive statistical results are presented as the median, 25th, and 75th percentiles. Comparisons between values before and 1 yr after surgery were performed using Wilcoxon’s matched pairs rank sum test, and comparisons between groups were carried out using the Mann-Whitney U test. Correlations were sought by calculating the Spearman rank correlation coefficient. A P-value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Non-22K GH isoforms in samples with different GH concentrations

Figure 1Go shows the proportion of non-22K GH isoforms found in peak, middle, and nadir samples from each acromegalic patient. A large spectrum of values was observed among patients, with the proportion of non-22K GH isoforms before and after surgery ranging from 14–51% and 8–62%, respectively. However, the values were fairly constant among different samples from the same patient, regardless of the GH level, both before and after surgery. Table 2Go shows the median values of total GH levels and the proportion of non-22K GH isoforms in peak, middle, and nadir samples, as well as the mean value of the samples in the study groups.



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  		Figure 1. The proportion of non-22K GH isoforms determined by the GHEA in different samples from acromegalic patients pre- (a) and 1-yr post transsphenoidal (b) surgery. The samples were selected from 24-h GH profiles and defined as follows: peak, maximum secretory GH peak; middle, level corresponding to the mean GH concentration observed during the 24-h period; nadir, lowest detectable GH level in the profile. In one patient, no peak and middle samples were available for measurements of non-22K GH isoforms after surgery, whereas two postsurgery middle samples were excluded because of undetectable levels of non-22K GH isoforms in the GHEA. Closed circles denote median values.

 

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  		Table 2. Total GH concentrations and proportion of non-22K GH isoforms in peak, middle, and nadir sample determined by the GHEA in the study groups

 
Non-22K GH isoforms in controls and acromegalic patients

Figure 2Go shows the proportion of non-22K GH isoforms observed in peak samples from acromegalics and controls. The values obtained in 13 normal men ranged from 15–38%. Patients with active acromegaly had an increased proportion of non-22K GH isoforms, in comparison with controls (median 26.6 vs. 17.4%; P < 0.01). As shown in Table 2Go, in the operated patients of groups A and B, GH concentrations were similar in peak and middle samples, with peak values comparable with those in the controls. However, the proportion of non-22K GH isoforms ranged from 9–56% in postsurgery samples. In group A, the amount of non-22K GH isoforms was increased, compared with group B (P < 0.05 for the peak and middle sample and P < 0.01 for the mean value of the samples). The median proportion of non-22K GH isoforms in group A was comparable with that observed in the acromegalics before treatment, whereas in the group B, the median was similar to that seen in the controls (Fig. 2Go and Table 2Go). The analysis of 24-h profiles showed that only one patient in group A (mean 24-h GH of 1 µg/L and mean non-22K GH isoforms of 22.4%) had restoration of normal circadian rhythm after surgery, whereas only one patient in group B (mean 24-h GH of 0.5 µg/L and mean non-22K GH isoforms of 10.6%) had an altered GH pattern, as defined in this study by the failure of baseline values to fall below 0.05 µg/L in the profile.



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  		Figure 2. Box plot of the proportion of non-22K GH isoforms determined in peak samples (maximum secretory GH in 24-h profile) obtained from normal men (n = 13, controls), untreated acromegalic men (n = 15, presurgery), and acromegalic patients in whom the mean 24-h GH concentration 1 yr after surgery was 1 µg/L or more (n = 6, group A) or less than 1 µg/L (n = 8, group B). The five horizontal lines display the 10th, 25th, 50th (median), 75th, and 90th percentiles (P < 0.05 presurgery and group A vs. control and group B vs. group A).

 
Correlations among non-22K GH isoforms, laboratory measurements, and body composition

The proportion of non-22K GH isoforms correlated directly with the mean 24-h GH concentration in the acromegalics both before (P = 0.01) and after surgery (P = 0.001). The amount of non-22K GH isoforms was not correlated with IGF-1 (IGF-1 SD scores), IGFBP-1, IGFBP-3, or insulin concentrations. Peak GH level and mean 24-h GH concentration were directly correlated with IGF-1 (IGF-1 SD scores) before surgery (P = 0.01 and 0.02, respectively), but such correlation was not observed post surgery (P = 0.66). The IGF-1 SD score did not normalize in three patients of group B, in whom mean 24-h GH was below 1 µg/L, whereas it was normal in one patient of group A. In untreated acromegalics, a positive correlation was found between serum PRL (P = 0.04) and ECW (P = 0.03) with non-22K GH isoforms. However, ECW also was correlated to mean 24-h GH concentration (P = 0.02). No difference in hormone measurements or body composition was found between patients of groups A and B, although insulin, IGFBP-3, ECW%, and BF% in group B and IGFBP-1 and IGFBP-3 in group A were different, compared with presurgery values (Table 1Go).

Correlations between non-22K GH isoforms and tumor size

The tumor size was correlated positively with the proportion of non-22K GH isoforms and serum PRL levels (P < 0.001). In one patient with a small adenoma (tumor size 0.06 cm3), a high proportion of circulating non-22K GH isoforms (peak sample, 32.4%) was observed. The amount of non-22K GH isoforms also was correlated directly with the grade of parasellar invasion of the tumor (P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our results show that acromegalic patients have a higher proportion of non-22K GH isoforms at GH peaks, in comparison with healthy subjects, as determined by the GHEA. This new immunomagnetic-extraction-based assay is a useful screening method to identify conditions in which the proportion of circulating non-22K GH isoforms is altered. The anti-22K MCB used in our assay was selected because of its high affinity and specificity to 22K GH (26, 33). In comparison with other methods, the GHEA is technically simple, has a high sample capacity, and allows the measurement of total GH and non-22K GH isoforms simultaneously. Moreover, its performance can be predicted at different serum GH concentrations by the use of statistical experimental designs, which were used for systematic optimization of all parameters involved in the serum extraction of 22K GH (26).

Several GH isoforms and fragments can be generated by pre- and posttranslational mechanisms (4). In normal subjects, Baumann et al. (34) showed that 22K GH is the most abundant isoform at GH peaks, whereas non-22K GH isoforms and immunoreactive fragments are prevalent in the basal state. In contrast, we observed that the proportion of non-22K GH isoforms was fairly constant in different samples of the same acromegalic subject, both before and after surgery, regardless of GH concentration. However, a wide range of values was observed among patients. After surgery, the proportion of non-22K GH isoforms ranged from 9–56%, and this variation was observed in samples with approximately the same GH concentration, confirming that the proportion of non-22K GH isoforms in individual samples was not dependent on GH levels. It is likely that the observed variability was caused by tumors of different size secreting variable amounts of non-22K GH, because the proportion of these isoforms in circulation was related directly to the size of the adenoma. In addition, patients with more invasive tumors also had an increased proportion of non-22K GH isoforms. In our group, there was one exception in which a microadenoma was associated with high proportion of non-22K GH isoforms in the circulation, indicating that certain small tumors also can secrete isoforms in increased amounts. In view of the current knowledge on the genetic basis of acromegaly (35), it is possible that different mutations produce tumor cells with a potential to secrete several native or aberrant isoforms in variable proportions. Unusual alternative splicing products of the human pituitary GH gene have been described in pituitary tumor tissue (36). Moreover, basal and stimulated GH levels, as well as tumor size, differ in patients whose pituitary tumors contain mutant Gs protein, compared with those without this mutation (37, 38). However, it remains to be elucidated whether the heterogeneous nature of GH secreted by pituitary adenomas has implications for the management of acromegalic patients.

The definition of cure in acromegaly long has been a matter of debate. Melmed et al. (35) recommended that a cured patient should exhibit return of GH secretion to a normal 24-h secretion rate and restoration of normal circadian rhythm. Studies on body composition by Bengtsson et al. (39) showed that ECW and BF normalized in patients with posttreatment GH levels below 5 mU/L. In addition, analysis of 24-h GH profiles has shown that some patients with serum GH below 2 µg/L after a glucose load and normal IGF-1 levels do not necessarily recover a normal GH secretory pattern (40). Thus, the serum GH level to be reached is still uncertain, and it has been modified from initial values of 5 µg/L to 2 µg/L (41), with some authors suggesting the use of 1 µg/L as the cut-off level (42). Interestingly, we observed that patients with a mean 24-h GH concentration of 1 µg/L or more had increased amounts of non-22K GH, compared with those with a mean less than 1 µg/L. The GH secretion pattern was normal in only one patient of the former group, whereas the pattern was normal in all but one patient of the latter group. These results show that more non-22K GH isoforms continue to be produced, possibly by tumor remnants, in patients who are not truly cured after surgery.

The role of serum IGF-1 levels as a good marker of the presence of GH hypersecretion in acromegaly is controversial (20, 42, 43). Our results showed that serum IGF-1 levels were correlated with peak GH and mean 24-h GH concentration in acromegalics before surgery, but a complete lack of correlation was found after surgery. Furthermore, we observed patients with normal mean 24-h GH concentrations and no abnormalities in the GH secretion pattern in whom IGF-1 SD scores were still altered. In this study, we have used age and sex-adjusted reference values for serum IGF-1 levels from a large reference population (29), and we believe that the lack of such conversion may be an explanation for discrepancies among studies. In addition, our results showing a very high variability in the proportion of non-22K GH isoforms in post-surgery samples, in which GH levels were present in a narrow range, suggest that differences in immunoreactivity and bioactivity among various non-22K GH isoforms partly explain the absence of correlation between mean 24-h GH concentration and IGF-1 levels observed in some patients.

In untreated acromegalics, the amount of non-22K GH isoforms was directly correlated to the mean 24-h GH concentration and serum PRL levels. Interference of PRL in our assay could be ruled out because GH levels and mean 24-h GH concentration were not correlated to PRL in this group. Hence, adenomas secreting more GH and PRL seem to secrete more non-22K GH isoforms, as well. The observed correlations between non-22K GH isoforms and mean 24-h GH concentration with ECW suggest that the circulating 22K/non-22K GH ratio may play a role in some abnormalities of body composition observed in patients with acromegaly (44).

In conclusion, we have shown that acromegalics have an increased proportion of circulating non-22K GH isoforms, regardless of GH level. The values are constant in an individual, but a large spectrum of values can be observed among patients. This variability suggests that different pituitary adenomas may secrete non-22K GH isoforms in variable amounts. Our observation in this group, that a higher proportion of non-22K GH isoforms was present in patients not truly cured after surgery, suggests that the evaluation of non-22K GH isoforms might be useful in the follow-up of acromegalic patients.


   Acknowledgments
 
The anti-22K GH MCB was kindly provided by Genentech Inc. We are indebted to Per-Arne Lundberg, Lars Hynsjö, Ingvar Bosaeus, Birgitta Sandrén, and Lena Wirén for excellent biochemical and technical support.


   Footnotes
 
1 This work was supported by grants from the Medical Faculty of Göteborg and the Swedish Medical Research Council (11285, 11502, 11331, 11576, 11621), and Emil and Wera Cornells Stiftelse. Back

Received October 28, 1996.

Revised January 17, 1997.

Accepted January 31, 1997.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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