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Original Studies |
Department of Growth and Reproduction (A.J., E.M.-L., S.A.P., J.M., N.E.S.), National University Hospital, 2100 Copenhagen; Department of Clinical Physiology and Nuclear Medicine (S.M., J.H.), and Department of Endocrinology (M.H.R.), Hvidovre Hospital, 2740 Hvidovre; Department of Biostatistics (T.S.), Panum Institute, 2200 Copenhage N; Department of Pediatrics (K.W.K.), Glostrup County Hospital, 2600 Glostrup; Diagnostics Systems Laboratories, Inc. (J.M.), Webster, Texas 77598; and Centre of Preventive Medicine (S.R.), Glostrup County Hospital, University of Copenhagen, 2600 Glostrup, Denmark
Address all correspondence and requests for reprints to: Anders Juul, M.D., Ph.D., Department of Growth and Reproduction, Rigshospitalet Section 5064, 9 Blegdamsvej, DK-2100 Copenhagen Ø, Denmark. E-mail: ajuul{at}post4.tele.dk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eight years after the existence of ALS was suggested, ALS binding was further characterized (5) and purified (6), and ALS quantification by RIA was reported in 1990 by Baxter (7). Until recently, this has been the only quantitative assay for the study of circulating ALS, although semiquantitative immunoblot assays have been used (2, 8, 9). Preliminary reports have demonstrated low serum ALS levels in GHD patients (7, 10, 11, 12), but comparison of ALS concentrations in GH-deficient individuals vs. carefully age- and gender-matched control subjects has not previously been done.
To examine the prerequisites for ALS as a diagnostic parameter, we have studied the hepatic production rate and diurnal variation of serum ALS in healthy subjects, with a newly developed ALS enzyme-linked immunoassay (13). Furthermore, we present circulating ALS levels in sera from 1158 healthy subjects to provide valid normative data for this assay; and, based on these values, we evaluated the diagnostic value of ALS, compared with other IGF-related parameters, in 108 young adults with childhood-onset GH-deficiency.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Healthy children, adolescents, and adults, n = 1158. Seven-hundred-seventy-eight children participated. Samples were drawn from 46 children, 0–5 yr old (43 boys) before herniotomy or circumcision. Healthy children in the 5–20 yr age range, from 4 different primary schools and one grammar school in the Copenhagen area, participated in the study. Children with acute or chronic diseases or on medication were excluded. All had heights and weights within the normal ranges of Danish children. Total IGF-I, free IGF-I, and IGFBP-3 levels have previously been published in these children (14, 15, 16). Three-hundred-eighty (202 male) adults participated. As part of a large cohort study (DAN-MONICA) in the Copenhagen area, serum was taken from 211 adults (40, 50, 60, or 70 yr old). Furthermore, 169 hospital employees and medical students participated as controls in the 20- to 40-yr age group. None had acute or chronic diseases, and none were on any medication (including oral contraceptives).
Production rate. Thirty subjects (18 males/12 females), 41–79 yr old [mean, 63.5 (11.9) yr], with no signs of liver disease, participated. Catherization and hemodynamic investigations were performed to exclude intestinal ischemia. The patients were studied after an overnight fast. A small indwelling polyethylene catheter was introduced in the femoral artery by the Seldinger technique. Catheterization of the hepatic vein was performed as described by Henriksen et al. (17). Under local anesthesia, a Cournand or Swan-Ganz catheter was guided to the localizations via the femoral route, under fluoroscopic control. Hepatic blood flow was determined by the indocyanin green constant infusion technique (18) in 19 of the subjects. Hepatic plasma flow (HPF) was calculated as hepatic blood flow multiplied by (1-hematocrit), and the mean HPF was used in the 11 subjects where only arteriovenous gradients were obtained. The net production of ALS was calculated as HPF x ALS concentration (hepatic vein-femoral artery).
Diurnal variation. eight healthy subjects (5 males), 20–36 yr old [mean age, 27.25 (5.6) yr], participated. Body mass indices ranged from 20.2–25.79 [mean, 22.6 (1.6)]. Blood samples were drawn at 20-min intervals from a heparinized iv catheter using a constant withdrawal pump. Three consecutive serum samples were pooled in one-hourly samples for hormone analyses.
Patients
One-hundred-eight patients, who were previously treated with GH during childhood, were included in this study. They were studied to evaluate consequences of childhood-onset GH deficiency in adults, as well as to select patients for adult GH-replacement therapy. Their GH secretion was reevaluated in adulthood by GH provocative testing and grouped according to their maximal GH response after oral clonidine, as either GH-deficient (peak GH below 7.5 µg/L (GHD; n = 79) or as having normal GH response (peak GH above 7.5 µg/L; n = 29), as previously described. Detailed clinical characteristics of these patients have been reported (19).
Blood sampling. Serum levels of ALS, total IGF-I and free IGF-I, and IGFBP-3 were determined on a basal blood sample from all 1158 individuals and were compared with the peak GH value during the GH provocative test in the 108 patients. IGF-II levels were determined in 252 healthy subjects only. Blood samples were drawn from an antecubital vein and centrifuged, and serum was stored at -20 C until analysis.
Analyses
ALS
ALS was determined by newly developed commercially available
enzyme-linked immunosorbent assay (Diagnostics Systems Laboratories, Inc.) (13). Standards ranged from 1.09–70
mg/L. In our hands, interassay coefficients of variation (n = 22)
were 20.4% (at 2.8 mg/mL) and 12.1% (at 17.6 mg/L), respectively.
Intraassay coefficients of variation (n = 20) were 8.6% (at 30.1
mg/L) and 7.4% (at 8.4 mg/mL), respectively.
Total IGF-I
Total IGF-I was determined by RIA, as previously described (14).
Briefly, serum was extracted by acid-ethanol and was cryoprecipitated
before analysis, to remove interfering IGFBPs. Inter- and intraassay
variation were less than 9% and 6%, respectively. Details regarding
determination of total IGF-I have been presented previously (14).
Free IGF-I
Free IGF-I was determined by an immunoradiometric assay
(Diagnostics Systems Laboratories, Inc., Webster, TX).
Briefly, this assay is a noncompetitive assay in which the analyte is
sandwiched between two antibodies. It is a direct assay of the
dissociable fraction of IGF-I. In our hands, sensitivity was 0.03
µg/L and inter- and intraassay variation were 9.9% and 10.1% (at
6.9 µg/L and 4.3 µg/L), respectively. Details regarding
determination of free IGF-I have been presented previously (16).
IGF-II
IGF-II was determined by an immunoradiometric assay
(Diagnostics Systems Laboratories, Inc.). Serum was
extracted with acid-ethanol before IGF-II determinations. Inter- and
intraassay coefficients of variation were 7.2% (at 416 µg/L) and
6.3% (at 427 µg/L), respectively, and sensitivity was 0.13 µg/L.
IGFBP-3 was determined by an RIA, previously described (20). Reagents
for the assay were obtained from Mediagnost GmbH (Tübingen,
Germany). Sensitivity was 0.29 µg/L (defined as 3 SD from
the mean of the zero standard). Inter- and intraassay coefficients of
variation were 10.7% and 2.4% (at bound-to-free ratios of 0.4–0.5),
respectively. Details regarding determination of IGFBP-3 have been
presented previously (15).
Statistics
Reference curves for mean ALS levels were obtained by smoothing the untransformed data, using local linear regression (21), because ALS exhibited a normal (Gaussian) distribution. Similarly 2.5, 16, 84, and 97.5 percentiles, corresponding to -1, -2, 1, and 2 SD, were obtained from smooth-variance estimates. From these curves, age- and gender-related z-scores (i.e. number of SDs from an age- and gender-related mean were calculated for each value. Correlations were analyzed using Pearsson’s test. To estimate the diurnal variation of the parameters, we fitted a random-effect model, where the random effect allowed for intraindividual variation. Sensitivity of the parameters was defined as the percentage of GHD patients with a value below -2 SD. Specificity was defined as the percentage of patients with a normal GH response who had a value above -2 SD. Predictive value of positive test was defined as percentage of values below -2 SD that represent GHD patients. Predictive value of negative test was defined as percentage of values above -2 SD that represent patients with normal GH response. Test accuracy was defined as the number of GHD patients with a subnormal value plus the number of patients with a normal GH response who had a normal value, divided by the total number of patients. Results are expressed as mean (SE), unless otherwise stated. A P value of less than 0.05 was considered statistically significant.
Ethical considerations
All subjects participated after giving informed consent according to the Helsinki II declaration. The study was approved by the local ethics committee for medical research in Copenhagen (approval nos. V.100.2086/91 (catheterization study), V.100.1680/90 (diurnal variation study), and V200.1996/90 (healthy children).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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ALS correlated with total IGF-I (r = 0.72, P
< 0.0001), IGF-II (r = 0.38, P < 0.001),
IGF-I+IGF-II (r = 0.72, P < 0.001), and IGFBP-3
(r = 0.75, P < 0.001) (Fig. 1
).
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We found no significant arteriovenous gradient over the liver and,
consequently, no measurable hepatosplanchnic release of IGF-I, and
IGFBP-3 by this technique, in our subjects with no signs of liver
disease. By contrast, we found a significant hepatic extraction of ALS
(Table 1).
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The mean individual variations of the four IGF-related parameters
are shown in Table 2. ALS, total IGF-I,
and IGFBP-3 (but not free IGF-I) serum levels showed significant
variation during the 24-h period, with significantly lower levels
during the night (Fig. 2).
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ALS serum levels increased during childhood, with highest values
in puberty. A 95% prediction interval is shown in Fig. 3 (Table 3). There was a significant difference in
ALS levels, according to gender, with females having significantly
higher ALS levels, compared with males, and with ALS levels increasing
1–2 yr earlier in girls, compared with boys (Fig. 2
). In adult males,
there was a significant decrease in ALS with increasing age (r =
-0.34, P < 0.00001), whereas ALS levels remained
constant with increasing age in adult females more than 30 yr old (Fig. 4).
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Serum ALS levels were below -2 SD in 57 out of 79
patients with GHD and above -2 SD in 22 out of 29 patients
with a normal GH response (Fig. 5).
Sensitivities and specificities for all IGF-related parameters are
given in Table 4. ALS correlated
significantly with peak GH levels (r = 0.53, P <
0.001) and with the number of additional pituitary hormone deficits
(P < 0.0001). In all 108 patients suspected of GHD,
ALS SDs correlated significantly with IGF-I SDs
(r = 0.79, P < 0.0001), free IGF-I
SDs (r = 0.72, P < 0.0001), and
IGFBP-3 SDs (r = 0.79, P <
0.0001).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGF-I is complexed in serum with IGFBP-3 and ALS, which form a stable circulating pool that regulates bioavailability of IGF-I to the tissues. ALS circulates in molar excess (approx 250 nmol/L) to IGFBP-3 (approximately 100 nmol/L) (7), which in turn, circulates in molar excess to IGF-I but, in equimolar concentrations, to the sum of IGF-I and IGF-II (15). We found that ALS levels were more closely related to IGFBP-3 levels than to those of the IGFs. This may reflect their coregulation in the liver, or the fact that all IGFBP-3 is bound to ALS and rapidly cleared from the circulation in its unbound form. Recently, it was shown that ALS bind to the IGFBP-3-IGF-I binary complex with a constant of 2.5 x 108 L/mol (24), which is 300-fold lower than the constants for the formation of the binary complex. It is likely that dissociation of the weak ALS-IGFBP-3 bond readily occurs at the endothelial surface mediated by cell-surface-associated glycosaminoglycans (25), hereby facilitating the IGF transport to the tissues. Despite their close and dynamic interactions, all three components of the ternary complex are regulated by GH and can, in the present context, be regarded as independent indicators of GH secretory status. However, within the liver, the biosynthesis of different proteins have been attributed to different cell types; that of IGF-I and ALS to the hepatocyte and that of IGFBP-3 to nonparenchymal cells (3). Consequently, they may vary in their regulation, dependence on nutritional status, and diagnostic utility.
We did not find any statistically significant arteriovenous gradient for IGF-I, or IGFBP-3, but to our surprise, we found a significant hepatic extraction of ALS for which we have no explanation. Estimation of the daily ALS production has not previously been attempted, but the IGF-I production rate has been estimated in other studies by different techniques. Kinetic studies in healthy subjects, as well as mathematical modeling, have estimated a total daily IGF-I production of approximately 3–10 mg/day (26, 27, 28, 29, 30, 31). If all circulating IGF-I was derived from the liver, and a hepatic plasma flow of 0.7 L/min was assumed, one would expect an hepatic arteriovenous gradient for IGF-I of 3–10 µg/L, which we and others (32, 33) were not able to detect. Based on animal and cellular experimental studies, circulating ALS is assumed to be derived from the liver. Hypophysectomy of rats decreases hepatic ALS gene transcription and messenger RNA levels (4). Conversely, GH administration increases rat hepatic ALS gene transcription and ALS promotor activity (34), ALS production from isolated hepatocytes (35), and circulating ALS levels in humans (10, 11, 12). By contrast, addition of IGF-I to isolated hepatocytes had no effect on ALS production (36), whereas sc administration of recombinant IGF-I to healthy subjects suppresses ALS (37), probably because of decreasing endogenous GH secretion. Altogether, these findings speak in favor of GH as the primary regulator of serum ALS levels. However, the hepatic origin of circulating ALS in humans has not been proven, so far; and furthermore, ALS has been demonstrated in extravascular fluids, such as peritonal, follicular, and synovial fluids (8, 13), which could argue for transcapillary transport of ALS, or local nonhepatic ALS production. In conclusion, no human in vivo study, including the present study, has been able to demonstrate a basal hepatic ALS, IGFBP-3, or IGF-I production in humans. This may be caused by methodological problems or, alternatively, the fact that the liver is not the primary source of circulating ALS, IGF-I, or IGFBP-3.
Diurnal variation of ALS has, to our knowledge, not previously been reported. We found a relatively small diurnal variation in circulating ALS levels, amounting to 12%, indicating that a single determination of this parameter is not subject to large variation and, therefore, may be suitable for screening of GH disorders. Diurnal variations of total IGF-I and IGFBP-3 were even smaller (amounting to 8% and 9%, respectively), whereas the mean individual diurnal variation for free, dissociable IGF-I was relatively high (32%). Two previous studies reported on decreasing IGF-I levels during that night (38) or increasing IGF-I levels during the night (39). These differences are probably caused by methodological differences in the IGF-I assays. We found a distinct diurnal variation for IGFBP-3, with significantly lower nighttime levels, in accordance with two previous reports (40, 41). However, we do not believe that the decreasing levels during night have any clinical implication, because the levels were constant from 0800–1400 h, when blood samples are usually drawn in an outpatient clinic.
The diagnostic value of ALS in patients suspected of GH insufficiency has not previously been systematically evaluated. We have previously shown that ALS levels are low in GHD adults and increase after GH replacement (12), in accordance with recent findings by others (10, 11), confirming the GH dependence of the peptide. In our present large cohort of 108 young adults with childhood-onset GHD, previously treated with pituitary-derived GH, who were reevaluated with GH provocative testing after cessation of GH treatment, we found that ALS levels predicted the outcome of GH provocative testing with a diagnostic accuracy of 73%, which may seem inappropriately low. However, GH provocative testing in adults, using clonidine as stimulus, is subject to large variability, because a large proportion of healthy individuals have a pathologically low response (42). Equally, a very large variation has been demonstrated for the insulin tolerance test (43), which is generally considered the optimal test to perform in adults (44). Therefore, one could argue that the suboptimal diagnostic accuracy of ALS, in terms of predicting the response to provocative testing, could disclose ALS determination, as well as provocative testing in adults, as diagnostic tools in adults with GHD. Similarly, IGF-I has been suggested to be a very poor discriminator in adult-onset GHD adults (45) but a reasonable diagnostic tool in patients with childhood-onset disease (19, 46). At present, diagnosing adults with GHD is still an enigma; and certainly, a nonphysiological GH provocation test, using arbitrary GH cut-off levels, is insufficient as a single test.
In conclusion, we have provided extensive normal data for a novel ALS assay and found that circulating ALS levels exhibit minor diurnal variation. We suggest that ALS determination may improve correct future classification of adults suspected of GHD, whereas the diagnostic use of ALS in short children remains to be evaluated.
Received May 14, 1998.
Revised August 13, 1998.
Accepted August 24, 1998.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) subunit of the high molecular
weights insulin-like growth factor-binding protein complex. J Clin
Endocrinol Metab. 70:1347–1353.[Abstract]
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7.5 µg/L), during a GH provocative test [male patients
(left figures) and female patients (right
figures)]. The lines represent mean and 95%
prediction interval in healthy subjects.