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The most important cause of hypoglycaemia in the presence of high insulin and C-peptide concentrations is insulinoma. However, a similar picture arises from use of sulphonylureas, which is sometimes covert. All specimens received in two years by a supraregional assay service laboratory from adults with low glucose and inappropriately high insulin and C-peptide concentrations were tested for sulphonylureas by a radioimmunoassay that employed antibodies to glibenclamide. In sulphonylurea-positive cases a questionnaire was sent to the consultant responsible for the patient, to elicit further information. Samples from 93 adult patients met the criteria, and 34 (37%) of these gave a positive result on screening for sulphonylureas. The consultants provided further information on 31 of the 34, and in 20 the presence of a sulphonylurea was unexpected. In 10 the features were such as to raise the possibility of factitious drug ingestion. A simple screening technique applied to specimens from patients with hyperinsulinaemic hypoglycaemia indicated that, in a substantial proportion of cases, the patient was taking a sulphonylurea.

Objective To assess the relative efficacy of hGH and glucocorticoids in the treatment of non-islet cell tumour hypoglycaemia (NICTH) by examination of their influence on the composition of the various molecular species involving tumour and mature forms of IGF-II in association with IGFBP-3. Design Two groups each of 4 patients, all diagnosed as cases of NICTH, were treated with either hGH or glucocorticoids. Through the use of acidic size exclusion chromatography serum levels of tumour (big) and mature IGF-II were evaluated. Neutral size exclusion chromatography was used in the separation of molecular species before assay for immunoreactive IGF-II and IGFBP-3 content. Results High-dose hGH treatment produced increases in serum levels of big and mature IGF-II and IGFBP-3 but without generation of high molecular weight complexes. Glucocorticoid treatment suppressed big IGF-II permitting re-establishment of normal IGF/IGFBP association patterns. Conclusion Glucocorticoid therapy has been demonstrated to consistently reverse the biochemical abnormalities caused by tumour-derived big IGF-II compared with the potentially adverse stimulatory effects of hGH treatment in causing increases in serum levels of big IGF-II.

We investigated whether pancreatic β-cell dysfunction has a role in the pathogenesis of glucose intolerance in acromegaly by comparing plasma intact proinsulin, immunoreactive insulin, C-peptide and glucose concentrations during a 75 g oral glucose load in six patients with active acromegaly and eight healthy volunteers. Only acromegalic patients with normal glucose tolerance were studied. Glucose concentrations were similar in acromegalic patients and controls. Acromegalic patients had higher fasting insulin ( P

OBJECTIVE While the effects of age on the growth hormone/insulin-like growth factor (IGF) axis are well documented, the influence of ethnic background is unknown. The differences in IGF and IGF binding proteins (IGFBPs) were investigated in two ethnic groups. DESIGN A cross-sectional study of an age-selected cohort of healthy, normoglycaemic, non-obese Caucasian (C) and Asian (A) subjects. PATIENTS Fifty-three (27 C, 26 A) subjects with a mean age (±SD) of 20.6±0.8 years were studied. MEASUREMENTS Fasting measurements of glucose, insulin, IGF-I, IGF-II, IGFBP-1 and IGFBP-3. Western ligand blotting and immunoblotting with IGFBP-2 and IGFBP-3 of serum samples. RESULTS There were no significant differences in IGF-I levels between Caucasian and Asian subjects (C 218±55 vs A 229±40μg/l; P=0.44). IGF-II (C 707±110 vs A 583±75μg/l; P

A patient presented with frequent episodes of spontaneous hypoglycaemia due to a solitary fibrous tumour of pleural origin, secreting incompletely processed proinsulin-like growth factor II (big IGF-II). Somatostatin receptors were demonstrated in the tumour by 111 In-labelled octreotide scintigraphy, but despite maximal doses of octreotide, there was no suppression of big IGF-II secretion and the hypoglycaemia persisted. The combination of GH and glucocorticoid therapy abolished the hypoglycaemia.

We have detected somatostatin receptors (SSR) by autoradiography in 3/4 established small cell lung cancer (SCLC) cell lines but not in two non-SCLC cell lines. The growth of 1/3 SSR positive SCLC cell lines was significantly inhibited by the long-acting somatostatin analogue octreotide (SMS 201-995, Sandostatin) 10(-9) M. We treated 20 SCLC patients with octreotide 250 micrograms three times daily for 1 week prechemotherapy (six patients) or at relapse after chemotherapy (14). Octreotide was well tolerated, and serum insulin-like growth factor-I levels were suppressed to 62 +/- 7% of pre-treatment levels. However there was no evidence of anti-tumour activity measured by tumour bulk or serum levels of neuron-specific enolase. In one patient metastatic skin nodules were shown to be SSR positive before and at the end of 2 weeks octreotide. Despite this the patient had progressive disease, and tumour cells obtained by fine needle aspirate before and after treatment showed no growth inhibition when cultured with octreotide immediately or following establishment as a cell line. In summary we saw little correlation between SSR expression and growth inhibition by octreotide, either in vitro or clinically. Images Figure 4

IGF-1 concentrations were measured in plasma from 20 obese patients and 20 age- and sex-matched control subjects all of whom were fasting. Plasma IGF-1 concentrations were lower (p less than 0.05) in the obese individuals than in the controls. Plasma GH and insulin responses to stimulation with oral glucose were measured in 7 healthy normal weight individuals and both before and after energy restriction in 7 obese individuals. Before treatment the obese individuals were hyperinsulinaemic (fasting and integrated stimulated concentrations p less than 0.05) and showed attenuation of the plasma GH response to stimulation (peak and integrated stimulated values p less than 0.05 and less than 0.005, respectively) following oral glucose when compared to normal weight controls. Plasma insulin levels fell (both fasting and integrated concentrations p less than 0.05 and less than 0.005, respectively) and stimulated plasma GH responses increased (both peak and integrated values p less than 0.01 and less than 0.05, respectively) in the obese individuals following 3 wk treatment with a synthetic very low energy diet. Plasma IGF-1 concentrations remained unchanged during energy restriction. These results fail to support an endocrine IGF-1 mediated feedback inhibition on the hypothalamic-pituitary axis as the cause of the attenuated release of GH in obesity.

Laron-type dwarfism, I a syndrome of growth hormone (GH) receptor deficiency, is an autosomal recessive disorder caused by a variable genetic defect of the GH receptor.s-' The phenotype of the patients with Laron-type dwarfism (LTD) resembles isolated GH deficiency." LTO is characterized by extremely low serum insulin-like growth factor-I (IGF-I) despite normal or high concentrations of biologically active GH and by resistance to exogenous GH. 5 The defect in the GH receptor results in deficiencies of IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3), the major serum carrier protein of IGF peptides.s? Purification and protein sequencing revealed that serum growth hormone binding protein (GHBP) was structurally identical to the extracellular hormone binding domain of the membrane-bound GH receptor." The GHBP was noted to be absent in some LTO patientsv'" and normal in others. II We present here an unusual case of GH receptor defect and the members of two unconnected families, one with three siblings with LTO and GHBP-negative phenotype and the other with one child with LTO and GHBPpositive phenotype.

Summary objective To compare the outcome of different treatment options used in several cases of non-islet cell tumour hypoglycaemia (NICTH). patients Eight cases of NICTH were referred for diagnosis and monitoring following either surgical or medical treatment. methods Serum samples collected throughout the time-course of each case were analysed for glucose, insulin, C-peptide, IGF-I, total IGF-II, total IGF-II to IGF-I ratio and, in most of the cases, big IGF-II. results Surgical excision was successful in the relief of symptoms and normalization of the biochemical parameters. Therapeutic treatment with glucocorticoids confirmed previous studies showing the suppressive effect on tumour (big) IGF-II production. The present data show that the effect was dose-dependent and reversible if doses were below a critical level. conclusions Within the limits of the cases studied, and the time-scales involved, moderate- to high-dose glucocorticoid therapy had immediate beneficial influence on symptomatic hypoglycaemia and, if tolerated in the long term, was effective in correcting the underlying biochemical dysfunction, unlike other therapeutic regimens. This effectiveness was only achieved when the dose exceeded a threshold level specific to the patient. In addition, reduction of the dose or withdrawal of the drug caused a return of the abnormal biochemical profile. Surgical removal of the malignancy, where this was an option, was successful within the periods studied.

Adult spontaneous hypoglycaemia is not a diagnosis per se but a manifestation of a disease. Although rare, it is important to identify spontaneous hypoglycaemia and its causes because treatment may be preventative or curative. Hypoglycaemia can occur as an epiphenomenon in many serious diseases. It is sufficient to recognise the disease’s association with hypoglycaemia and then take appropriate action to prevent the recurrence of hypoglycaemia. In investigating apparently healthy individuals, common pitfalls to avoid are: failure to recognise subacute neuroglycopenia clinically; failure to document hypoglycaemia adequately during symptoms; failure to measure pancreatic hormones, counter-regulatory hormones, and ketones in hypoglycaemic samples; failure to recognise pre-analytical and analytical limitations of laboratory assays; and failure to abandon obsolete and inappropriate investigations. Providing these caveats are met, appropriate laboratory and radiological investigations will almost always uncover the cause of spontaneous hypoglycaemia.

Aim: To assess the extent to which biochemical analytical services contribute to the diagnosis and management of clinical cases of hypoglycaemia. Methods: All cases of confirmed hypoglycaemia, referred during a six month period, were included in the survey. Questionnaires were sent to each referring laboratory requesting information on the clinical progress and current status of the patient. Results: The level of influence exerted by analytical data was assigned in each case and those with similar outcomes combined. Identifiable case groups were: (1) Results not recorded in the patients' notes (15.7%). (2) Inappropriate requesting of insulin and C peptide measurements in cases of diabetes (11.4%). (3) Patient died soon after investigation (20.0%). (4) Patient recovered spontaneously (17.1%). (5) Patient received effective medical or surgical treatment (12.9%). (6) Patient awaiting or not requiring pathology based treatment (31.4%). (7) Inconclusive outcome prompting further investigation (5.7%). Conclusions: Within the timescale of the survey (approximately 12 months), positive progress had been made towards diagnosis and subsequent treatment in only 10% of cases. Another 30% were either awaiting some form of treatment or further diagnostic tests. The remaining 60% did not appear to benefit in any way from the biochemical investigations.

Hypoglycemia occurs as an epiphenomenon in many serious diseases and further investigation may be unnecessary. In other, often seemingly healthy individuals, it is responsible for their presenting symptoms. In them preliminary diagnosis depends on demonstrating a low blood glucose concentration during spontaneous symptoms by ambulatory self-collection of capillary blood and its analysis for glucose in the laboratory. Subsequent investigation requires appropriate plasma hormone analysis on blood collected while the patient is hypoglycemic.

Hypoglycemia is a condition commonly seen in the emergency department and is usually caused by insulin or sulfonylurea therapy for diabetes mellitus. Tumor-induced hypoglycemia occurs more rarely and can involve several mechanisms, according to whether the tumor is of pancreatic islet-cell origin or of extrapancreatic, non–islet-cell origin.1 The pancreatic islet beta-cell tumors (insulinomas) cause hypoglycemia by producing excessive insulin. In contrast, non–islet-cell tumors can cause hypoglycemia in any of several ways. They include release by the tumor of insulin-like growth factor II or its high-molecular-weight precursor,2–4 multiple metastases to the liver, massive tumor burden, or rarely, the production of . . .

To assess the relative efficacy of hGH and glucocorticoids in the treatment of non-islet cell tumour hypoglycaemia (NICTH) by examination of their influence on the composition of the various molecular species involving tumour and mature forms of IGF-II in association with IGFBP-3.Two groups each of 4 patients, all diagnosed as cases of NICTH, were treated with either hGH or glucocorticoids. Through the use of acidic size exclusion chromatography serum levels of tumour (big) and mature IGF-II were evaluated. Neutral size exclusion chromatography was used in the separation of molecular species before assay for immunoreactive IGF-II and IGFBP-3 content.High-dose hGH treatment produced increases in serum levels of big and mature IGF-II and IGFBP-3 but without generation of high molecular weight complexes. Glucocorticoid treatment suppressed big IGF-II permitting re-establishment of normal IGF/IGFBP association patterns.Glucocorticoid therapy has been demonstrated to consistently reverse the biochemical abnormalities caused by tumour-derived big IGF-II compared with the potentially adverse stimulatory effects of hGH treatment in causing increases in serum levels of big IGF-II.

OBJECTIVE IGF-I and IGF binding protein (IGFBP)-3 levels in man are positively regulated by GH status; in contrast, evidence suggests an inverse relationship between GH status and IGFBP-2. We investigated the effects of somatropin administration on the serum concentrations of these analytes, together with serum and urinary concentrations of GH, to evaluate their potential as markers in the development of a test for detecting doping with GH in sports competitors. DESIGN Somatropin was administered subcutaneously at a dose of 0.15 U/kg bodyweight/day at 1000 h for 3 days to eight healthy men (20–32 years old). MEASUREMENTS Serum concentrations of GH, IGF-I, IGFBP-2 and -3 were determined in blood samples collected at 1600 h on the days prior to (day −1), during (days 0, 1 and 2), and following administration (days 3 and 7). Urine was collected continuously from days −2 to 3 and then on day 7. RESULTS Serum and urinary concentrations of GH were only raised on the days of administration whereas, following cessation of somatropin, the increases in the serum concentrations of IGF-I and IGFBP-3 were sustained for at least 1 day (30 h). Serum IGFBP-2 decreased during the period of administration and was still suppressed on day 3. The concentration ratios of IGFBP-3 to IGFBP-2 and IGF-I to IGFBP-2 increased markedly with administration and both ratios were still significantly augmented compared with basal values 30 h after the last administration. CONCLUSION With acute administration of somatropin to healthy men the serum concentration of IGFBP-2 decreases and the ratios of serum IGF-I/IGFBP-2 and IGFBP-3/IGFBP-2 increase. These ratios should be considered in the development of a test for detecting somatropin administration in sport.

Selected combinations of tests for growth hormone and the growth mediating peptides, insulin-like growth factors, and their binding proteins have improved the diagnostic reliability of the procedures used in the investigation of growth abnormalities (GH deficiency, GH and IGF receptor deficiencies, GH excess, diabetes, or undernutrition) and in malignancies associated with hypoglycemia.

Octreotide, a long-acting somatostatin analogue, has been used to alleviate hypoglycaemia in patients with insulinomas. Transient worsening of fasting hypoglycaemia following octreotide has also been described (Stehouwer et al., 1989). We describe a patient with a 'proinsulinoma' in whom octreotide caused rapid and prolonged symptomatic worsening of fasting hypoglycaemia. Catecholamine and cortisol counterregulatory hormonal responses were normal but those of glucagon and GH were impaired. Acute neuroglycopaenic symptoms were present during octreotide induced hypoglycaemia, which was accompanied by and attributed to an acute reduction in pancreatic glucagon secretion in the presence of persistent and unsuppressed hyperproinsulinaemia. This suggests that glucagon may be important in maintaining glucose homeostasis in chronic hypoglycaemia due to endogenous hyperinsulinism even though its concentration in the peripheral blood is not raised.

We describe a case of recurrent hypoglycaemia associated with a hepatoma. During hypoglycaemia serum insulin was undetectable. Plasma insulin-like growth factor II (IGF-II) was not elevated although 71% of plasma IGF-II was present as big IGF-II (molecular weight 11 kDa) which probably represents a non-glycated form of pro-IGF-II. The GH response to hypoglycaemia was impaired and plasma levels of both IGF-I and the GH-dependent IGF binding protein (IGFBP-3) were low. A recently described unextracted assay directed against the first 21 amino acids of the E-domain (E-21) of proinsulin-like growth factor-II (pro-IGF-II) allows direct plasma estimation (plasma E-21) of larger molecular forms of IGF-II without interference from normal IGF-II and IGF binding proteins. Basal values were grossly elevated (23.7 and 23.8 nmol/l). Treatment with GH led to an increase in the mean plasma glucose across 24 hours (4.25 +/- 0.21 mol/l (mean +/- SEM) before treatment, compared with 4.86 mmol/l +/- 0.17 following GH (P < 0.01)) and a reduction in hypoglycaemic attacks. The treatment was associated with a rise in IGFBP-3 and small increases in insulin like growth factors. Subsequent treatment with the somatostatin analogue octreotide did not produce a significant change in plasma glucose levels or insulin-like growth factors. Two courses of intrahepatic adriamycin restored elevated levels of E-21 to normal. Total IGF-II remained normal and IGF-I increased. GH treatment was successfully withdrawn with no effect on plasma glucose or growth factor levels. The patient remained free from hypoglycaemia.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE--To investigate the predictive value of fasting insulin concentrations for subsequent fatal or non-fatal ischaemic heart disease at five year follow up and to examine the associations between insulin and other indicators of risk. DESIGN--A prospective population study among 2512 men aged 45 to 59 at recruitment. SETTING--A whole population sample of men resident in Caerphilly, South Wales. MEASUREMENTS--At recruitment fasting blood samples were taken for measurement of plasma lipids and serum insulin. Men were re-examined at a five year follow up and ischaemic heart disease events during this period were assessed from hospital notes, death certificates, and electrocardiograms. MAIN RESULTS--Diabetic men and those men with a fasting blood glucose of > or = 8 mmol/l were excluded from all analyses. In a univariate analysis the incidence of ischaemic heart disease increased with increasing concentration of fasting insulin, such that for men in the top 20% of the insulin distribution the odds of developing ischaemic heart disease were 1.87 relative to men in the bottom 20%. On multivariate analysis this relation disappeared on adjusting for plasma triglycerides, body mass index, and evidence of ischaemic heart disease at recruitment. CONCLUSION--In this population in South Wales there was no evidence that the fasting insulin concentration is an independent risk factor for ischaemic heart disease. The univariate association between insulin and incident disease was almost entirely explained by the association of both with triglycerides and body mass index.

OBJECTIVES: Insulin related growth factor 1 (IGF1) is an important growth promoting peptide which has anabolic effects on in vitro cartilage metabolism. The fact that serum levels tend to decrease in elderly subjects while osteoarthritis (OA) increases in prevalence suggests that suboptimal levels may play a part in the development of OA. This study therefore considers the hypothesis that IGF1 levels are low in subjects with OA of the knee. METHODS: Serum IGF1 levels in a community derived sample with symptomatic knee OA were compared with those in normal controls matched by age and gender. Overall radiographic score, osteophyte score, sclerosis score, and joint space narrowing score were correlated with IFG1 levels. RESULTS: One hundred and seventy-seven elderly subjects (83 with knee pain, 94 without knee pain) participated, allowing the identification of 78 case-control pairs matched by age and gender (mean 71.8 v 71.9 years, SD 7.6 for the two groups). No difference was found in serum IGF1 levels between the cases and controls (mean(SD) 15.9(4.7) nmol/l, for cases, 16.4(5.4) for controls). Significant negative correlations were found between IGF1 levels and age, total radiographic score, osteophyte score, and sclerosis score. In a multiple regression model, however, only age remained significant. CONCLUSIONS: This study provides no evidence that serum IGF1 levels are low in OA. Apparent correlations with radiographic features of OA may be due to the confounding effects of age.

Tamoxifen treatment of women with advanced breast cancer has previously been reported to reduce plasma insulin-like growth factor-type I (IGF-I) concentrations. In this study we have examined the effect of treatment with Tamoxifen, medroxyprogesterone acetate (MPA) or 4-hydroxyandrostenedione (4-OHA) on plasma IGF-I and IGF-II concentrations. As IGF-binding proteins (IGFBPs) can modulate the biological effects of IGF-I, plasma IGFBP-I levels were also measured. Treatment with Tamoxifen for 2 weeks resulted in a small, but significant, decrease in IGF-I levels, but increase in the plasma concentration of IGFBP-I. In contrast, treatment with MPA increased levels of IGF-I, but significantly reduced plasma IGFBP-I concentrations. Treatment with 4-OHA had no significant overall effect on plasma IGF-I or IGFBP-I levels, although changes were detected for some subjects. Plasma IGF-II concentrations were not altered by treatment with Tamoxifen, MPA or 4-OHA. It is concluded that although treatment with Tamoxifen or MPA produced significant changes in plasma IGF-I concentrations, any physiological effects of such changes are likely to be modulated by the corresponding alterations in plasma IGFBP-I concentrations. © 1992 Wiley-Liss, Inc.

Spontaneous hypoglycaemia was the presenting feature of a man with a large subpleural fibroma. Preoperative and postoperative studies support the view that the tumour induced hypoglycaemia was due to the secretion of peptide hormones by the tumour.

We showed previously that insulin-like growth factor I (IGF-I) is detectable in small cell lung cancer (SCLC) tumor biopsies and cell lines and that recombinant human IGF-I stimulates DNA synthesis in SCLC cells. Here we report further studies on the role of IGF-I in 2 SCLC cell lines: HC12, classic; and ICR-SC17, variant. Immunoreactive IGF-I was detected in medium conditioned by HC12 but not ICR-SC17. Both HC12 and ICR-SC17 bound IGF-I with 100-fold greater affinity than insulin. Scatchard analysis revealed two classes of IGF-I binding site of high (Kd 0.1 nM, n = 2,300) and lower (Kd 3 nM, n = 28,000) affinity. In both cell lines [3H]thymidine incorporation was enhanced by recombinant human IGF-I, 100-1000 ng/ml. ICR-SC17 also showed growth enhancement as measured by increase in cell numbers. There was no response in HC12, probably due to endogenous IGF-I production. 125I-IGF-I binding and basal and IGF-I-stimulated mitogenesis were inhibited by monoclonal antibodies to IGF-I (SM1.20B, SM1.25) or the type I IGF receptor alpha IR3 but not an isotypic control monoclonal antibody. Antiproliferative effects were manifest in [3H]thymidine incorporation assays in serum-free conditions and growth of serum-supplemented liquid cultures. We also tested fresh or newly cultured tumor cells obtained by fine needle aspiration of metastases in three previously untreated and four relapsed patients with SCLC. IGF-I binding sites were demonstrable on fresh SCLC cells, and specific binding was inhibited by SM1.20B. All seven samples showed stimulation of [3H]thymidine incorporation in the presence of recombinant human IGF-I, 100-500 ng/ml. As in cultured cells, basal and IGF-I-stimulated DNA synthesis was inhibited by monoclonal antibodies SM1.20B, SM1.25, and alpha IR3 but not the isotypic control. These results confirm the findings of previous studies and suggest that IGF-I can function as an autocrine growth factor in SCLC in vitro and possibly also in vivo.

We investigated 24 hour growth hormone secretion by intermittent 20 minute blood sampling in 34 prepubertal patients with Turner's syndrome, aged 4.3-12.4 years. Growth hormone profiles were analysed by the PULSAR programme and results expressed as the sum of growth hormone pulse amplitudes. Six patients had abnormal growth hormone pulse frequencies. In the remaining 28, growth hormone pulse amplitudes declined significantly with increasing age, but there was no correlation between growth hormone pulse amplitudes and growth rates. Concentrations of insulin like growth factor-1 (IGF-1) rose with age but did not correlate with either growth rates or growth hormone secretion. Fifteen patients were given oxandrolone and 11 low dose ethinyl oestradiol. Both agents increased height velocity without increasing growth hormone secretion. We conclude that the relation between growth hormone secretion and growth in Turner's syndrome is less certain than in normal children. End organ resistance is probably due to a skeletal dysplasia. Both oxandrolone and low dose ethinyl oestradiol improve the growth of girls with Turner's syndrome, but their mechanism of action remains uncertain.

We have developed methods for measuring the concentrations of free GH in plasma using a polyethylene glycol (PEG) separation procedure to remove antibody-bound GH within 1 h of collection. Total GH concentrations were obtained by acidification of the GH–antibody complex to release the GH followed by PEG precipitation of the antibody. The plasma GH assay had a within-assay coefficient of variation (C.V.) of 6·8% at 4·6 mU/l and a between-assay C.V. of 9·2% at 4·0 mU/l. The PEG-modified assay had a within-assay C.V. of 4·3% at 6·3 mU/l and a between-assay C.V. of 10·9% at 5·3 mU/l. Both assays had a sensitivity of 1·3 mU/l. There was good correlation between plasma and free GH concentrations in 24-h profiles in two tall children (r = 0·98; P < 0·001) and between total and free GH in the same profiles (r = 0·97; P < 0·001). GH antibodies were measured using a highly sensitive radioimmunoassay. In children who did not develop GH antibodies there was no difference between total, plasma and free GH concentrations. In contrast, in those who developed GH antibodies both total and plasma GH concentrations were markedly increased compared with free GH concentrations. The presence of GH antibodies did not affect the growth, plasma insulin-like growth factor-I concentrations or fasting serum insulin concentration responses to 1 year of therapy with biosynthetic human GH. Journal of Endocrinology (1989) 121, 193–199

Recent developments in the production of antisera for use in immunoassays are discussed and their incorporation into drug radioimmunoassay techniques are reviewed. The contribution of the results from such assays to our knowledge of the pharmacokinetics and metabolism of particular groups of compounds is evaluated.

Antibodies, for use in radioimmunoassay, have been raised in sheep by immunization with a conjugate of Δ9-tetrahydrocannabinol hemisuccinate and bovine serum albumin. Antiserum titre and avidity were increased by successive booster doses of conjugate. The high degree of non-specific binding encountered in the radioimmunoassay of cannabinoids was reduced by the use of the solubilizing detergent Triton X-405 and by restricting protein concentration in the assay medium. Plasma samples were deproteinized with ethanol before assay, but urine was directly assayed. High avidity antibodies and high specific activity [3H]- Δ9-tetrahydrocannabinol permitted the detection of 50 pg of cross-reacting cannabinoids–-a sensitivity of 7.5 ng ml−1 of plasma and 1.0 ng ml−1 of urine. Whilst apparently specific for the three-ringed cannabinoid nucleus, the assay antiserum cross-reacted with several cannabinoids, both natural compounds and metabolites. Partial identification of cross-reacting cannabinoids was achieved by the use of pure compounds and by the assay of plasma and urine samples collected from rabbits given pure cannabinoids intravenously.

Of the somatomedins so far measured, the selective quantitation of insulin-like growth factor I (IGF-I) appears to have the greatest potential in clinical diagnosis. There have been two approaches to the development of immunoassay systems. One type uses antibodies raised against synthetic fragments of IGF-I which exhibit cross-reactivity with the whole hormone. Such assay systems may be adequate for measuring normal adult plasma IGF-I levels, but the potential for the higher sensitivity required for detecting sub-normal plasma levels in young children is apparent only in methods using antibodies raised against the complete hormone.IGF-I in plasma exists as part of a high molecular weight complex in which it is bound to carrier proteins. The binding proteins may interfere with plasma IGF-I measurements by radioligand assays. Direct analysis of untreated plasma samples is claimed to be possible using disequilibrium assay conditions but in order to maximise assay sensitivity it is necessary to employ an initial extraction stage in order to eliminate binding protein interference.Whether the measurement of plasma IGF-I can or should be used in addition to, or as a replacement for, plasma growth hormone (GH) measurement in the clinical assessment of growth disorders remains a controversial issue. Available evidence indicates that a single, random plasma IGF-I level provides an accurate reflection of GH secretion. Adequate discrimination between the elevated levels in acromegaly and normal reference values has been demonstrated. However, in the investigation of growth-retarded children available radioimmunoassay (RIA) methods have proved only partially successful because of the age-related nature of normal plasma IGF-I concentrations. Existing assays appear capable of identifying sub-normal plasma levels after the age of approximately 4 years. In younger subjects an improvement in assay sensitivity is required in order to establish with greater accuracy the relevant normal ranges.Improvements in the identification of the particular lesion responsible for retarded growth in a child can be achieved by measurement of both plasma GH and IGF-I concentrations. The predictive value of the acute plasma IGF-I response to single-dose GH therapy may identify patients who will respond to long-term GH therapy. Better, more informed decisions on subsequent treatment may therefore be made.Apart from GH control, several other factors influence circulating IGF-I levels. Nutritional status can be assessed through reference to IGF-I analysis, overall catabolic or anabolic processes being associated with decreasing or increasing plasma IGF-I levels respectively.

1 The cytotoxic agent, vinblastine, was conjugated to albumin, using the Mannich reaction. Rabbits immunized with two conjugates, containing differing amounts of hapten, produce antibodies which bound [3H]-vinblastine. 2 Antisera from one rabbit cross-reacted with both vinblastine and vincristine and were used to develop radioimmunoassays for measuring their concentration in plasma. 3 The antisera showed no cross-reactivity with other alkaloids or cytotoxic drugs and provided assays sensitive to a concentration of 2.1 ng vinblastine or 3.8 ng vincristine/ml of plasma added direct to the assay tubes. 4 This is sufficiently sensitive to permit the measurement of plasma vinblastine levels for up to 24 h after the intravenous administration of 15 mg of the drug.

Sixty four patients, age range 1-20 years, with cystic fibrosis had their tolerance to glucose assessed according to their glycosylated haemoglobin (HbA1) concentrations. Raised concentrations were found in 24 (37.5%). Oral glucose tolerance tests were performed on 21 patients with raised HbA1 and 13 patients with normal HbA1 concentrations. C peptide responses were also measured to assess islet cell function. Patients with normal HbA1 had normal glucose tolerance and C peptide response. Seven of 21 patients with raised HbA1 concentrations were glucose intolerant. The remaining 14 patients with raised HbA1 concentrations had normal glucose tolerance but a reduced C peptide response, suggesting impaired islet cell function. There were no appreciable differences in the incidence of chest infections, respiratory function, and chest x-ray scores among patients with normal HbA1 concentrations, raised HbA1 concentrations, and normal oral glucose tolerant tests, and patients who were glucose intolerant. No correlation was found between HbA1 concentration and age or Shwachman score. Measuring HbA1 concentrations periodically is useful in detecting and monitoring glucose intolerance in patients with cystic fibrosis.

Antibodies to bleomycin were raised by immunization of sheep and rabbits with bleomycin-albumin conjugates. The combination of a high-titre, high-avidity sheep antiserum and iodinated bleomycin produced a radioimmunoassay sensitive to 8 ng of bleomycin per ml of plasma or urine. Untreated specimens (100 microliter) of plasma or urine could be added directly to the assay tubes. The antiseerum was specific for bleomycin and showed no cross-reaction with other anticancer agents used in combination chemotherapy. Over a concentration range of 20-100 ng/ml, recovery of bleomycin from plasma was 110% and from urine, 93%. Repeated assay of plasma samples showed a decrease in bleomycin levels unless the samples were kept at 4 degrees C or below. Assay of bleomycin levels in plasma and urine from patients under treatment with bleomycin showed similarities with results reported using a microbiological assay. The radioimmunoassay offers a more reliable, rapid and sensitive method for the measurement of bleomycin.

A radioimmunoassay (R.I.A.), using a sheep antiserum and 3 H-T.H.C., has been developed for the measurement of tetrahydrocannabinol (T.H.C.) in plasma and urine. The antiserum cross-reacts with T.H.C. metabolites, but nevertheless possesses sufficient group specificity and sensitivity to permit measurement of cannabinoids in plasma and urine of animals and volunteers following the administration of pure T.H.C. Subjective effects experienced by the volunteers seemed to correlate with plasma-T.H.C. levels. The examination of random plasma urine samples collected from patients attending a drug-addiction clinic revealed the presence of measurable amounts of cannabinoids in several of them.

Circulating IGF-l concentrations are diagnosti­ cally raised in patients with active acromegaly even before non-suppression of growth hormone (GH) in response to oral glucose becomes evi­ dent.l" It has been suggested, therefore, that a single random serum IGF-l measurement could replace lengthy GH suppression tests in the diagnosis of agromegaly.'

The introduction of radioimmunoassay (RIA) by Yalow and Berson 15 years ago, for measuring first the insulin levels in plasma [18] and later those of other polypeptide hormones, led to its gradual application to almost every type of compound in biological fluids. Its use for measuring drugs has recently been reviewed [3, 4, 9].

Summary A radioimmunoassay for cannabinoids in blood and urine using an antiserum raised in sheep and [3H]-THC as label has been developed and used routinely in our laboratory during the past four years. It has proved itself useful clinically in the elucidation of obscure psychiatric disorders due to cannabis intoxication, in the study of cannabis bioavailability, the epidemiology of illicit cannabis use and its association with serious motor accidents.

An antiserum raised in sheep against a conjugate of tetrahydrocannabinol with bovine serum albumin has been used as the basis of a radioimmunoassay for cannabinoids in the blood of rabbits given tetrahydrocannabinol, 11-hydroxy-tetrahydrocannabinol, cannabinol or cannabidiol by rapid intravenous injection. In the case of both 11-hydroxy-tetrahydrocannabinol and cannabinol plasma cannabinoid concentrations fell exponentially from an initial peak plasma level attained immediately after the completion of intravascular distribution of the injected bolus. In the case of tetrahydrocannabinol itself, however, there was a progressive rise in plasma cannabinoid concentration between five and fifteen minutes after the rapid intravenous injection. The reasons for this rise in plasma cannabinoid concentration are discussed.

SynopsisThis study of 111 institutionalised Down's syndrome subjects and 74 controls found a relatively low risk of hypothyroidism in both groups compared with certain other studies of Down's subjects, which found very high rates of thyroid hypofunction. However, the Down's syndrome group had a significantly raised risk of hypothyroidism (9%), hyperthyroidism (1·8%) and the presence of thyroid microsomal antibodies (29%) compared with the controls and with laboratory reference data. It is recommended that thyroid function testing should be routine in older Down's syndrome subjects, with special attention being given to the clinical status of those with microsomal antibodies present.