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We have compared the stimulation of sulfate and thymidine uptake into 10-day-old embryonic chick cartilage by normal human serum, partially purified somatomedins (Sm) A and B, homogeneous insulin-like growth factors (IGFs) I and II, and several other substances. With the exception of epidermal growth factor, all growth factors ((GFs) were assayed in the absence of other protein. Pelvic rudiments were preincubated in buffer for 6 h and then incubated for 24 h with the GF or serum, with labels added for the final 6 h. Human serum enhanced cartilage uptake of both thymidine and sulfate. There was a dose-dependent stimulation of thymidine uptake by Sm A or B (0.05--2 microgram/ml) and IGF I or II (0.5--20 ng/ml). Unlike serum, neither Sms nor IGFs increased SO4 uptake under these conditions. Bovine GH (10--500 ng/ml), albumin (100-1000 ng/ml), fibroblast GF (1--100 ng/ml), and epidermal GF (1--100 ng/ml) were inactive for both thymidine and sulfate. When a shorter incubation was used (7 h), Sm A enhanced SO4 uptake, and discrimination was increased by preincubation of the rudiments in buffer for 24 h. With this procedure, IGF I (0.5 ng/ml) was nearly equipotent to 5% serum. On a weight basis, IGF I was more active than either Sm A or IGF II. The data suggest that assay conditions are crucial for demonstration of Sm activity. Appropriate conditions may be different for isolated GF than for a complex medium such as serum. The results further suggest that with certain protocols, the responsiveness of chick embryo cartilage is qualitatively similar to that of hypophysectomized rat cartilage.

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The embryonic chicken cartilage somatomedin bioassay was modified so that human serum stimulation of simultaneous [3H]methylthymidine and H2[35S]O4 incorporation could be assessed. The assay consisted of a 6 h pre-incubation of 10 day pelvic rudiments in enriched buffer, followed by a 24 h incubation with buffer and low (0.5, 2 and 5% v/v) serum concentrations. Both labels were present for the final 6 h. Other modifications were shortening of washing, elimination of drying and weighing, and simplification of digestion. Normal human serum produced a linear log dose-response with these serum concentrations. Potency ratios in patients with GH deficiency were less than those of normal adults for both thymidine 0.39 +/- 0.05 (mean +/- SEM, n = 16, range 0.22-0.71) vs. 0.90 +/- 0.05 (n = 19, 0.62-1.36, P < 0.001) and for sulphate 0.40 +/- 0.04 (0.15-0.65) vs. 94 +/- 0.05 (0.61-1.29, P < 0.001). Potency ratios for both labels rose following administration of a single dose (0.2 IU/kg im) of hGH to 4 GH deficient children. The reliability of prediction of GH deficiency, reproducibility, and precision were similar to other Sm bioassays. The major advantages of these modifications were the ability to examine 2 cartilage metabolic processes simultaneously and the small amount of serum (350 mul) necessary for patient assays.

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A 51-yr-old, nonobese, male patient presented with hyperglycemia and a recent 40-pound weight loss. Severe insulin resistance was documented in studies in which high amounts of insulin were infused using the Biostator GCIIS. Diabetic control was finally achieved with subcutaneous injections of 470 U of insulin per day. Positive laboratory findings included a mild pancytopenia, elevated erythrocyte sedimentation rate, decreased C3 and properdin, and increased IgA. Antinuclear or other autoantibodies were not present. Insulin antibody levels were within the range usually present in insulin-treated diabetic patients. Acanthosis nigricans was not present. Incubation of the patient's serum with IM-9 lymphoblastoid cells revealed that an insulin receptor antibody was present in a serum dilution of 1:80. Insulin-resistant diabetes mediated by insulin receptor antibodies may present in patients with immunologic findings but without overt dermatologic stigmata.

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The stimulation of isolated chicken embryo chondrocytes was studied by measuring the incorporation of [3H]uridine and [3H]leucine into cold trichloroacetic acid precipitable material after exposure of the chondrocytes to serum. The doseresponse relationships for the incorporation of uridine and leucine were similar to that of thymidine previously demonstrated. Exposure of the cells to serum-containing buffer for 15 min sufficed both for the stimulation of incorporation into the cells and for the depletion of 28% of the stimulating activity from the medium. Stimulation persisted for at least 17 h after removal of the serum. Studies where actinomycin D was added to inhibit RNA synthesis suggested that prior RNA synthesis was required for most of the stimulation of protein synthesis by serum factors.

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The effects of GH on cartilage may be mediated by a variety of serum factors (somatomedins; SM). We have reported (Endocrinology 90: 1086, 1972) stimulation of thymidine incorporation in isolated chicken embryo chondrocytes by normal human serum. This was greater than that caused by serum from patients with hypopituitarism. We have now compared the stimulatory activity estimated by [3H]thymidine incorporation (SMT) with that estimated by [3H]leucine incorporation in 46 sera from children with GH deficiency; with short stature, but normal GH responsiveness; or with normal stature and normal GH responsiveness. These activities were also measured in sera from 9 normal adults and 12 acromegalics. Sera from GH deficient children had reduced SMT activity (.54 +/- .04; (mean +/- SE) P less than .01) compared to normal children (.83 +/- .08) whereas the sera from children with short stature and normal GH responsiveness had higher levels than normal (1.19 +/- .10: P less than .02). Acromegalic adults averaged higher SMT activity than normal adults (1.62 +/- .15 vs. 1.17 +/- .11; P less than .05). In sharp contrast, the leucine incorporation was essentially the same in the different groups of children. These studies have validated the use of the incorporation of thymidine into isolated chicken embryo chondrocytes as an adjunct in the evaluation of children with short stature (82.6% of the samples from children gave results that were consistent with their status as determined by provocative tests for GH). The disparity between the results with thymidine incorporation and those with leucine incorporation is as yet unexplained.

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The hepatic radioreceptor assay for hGH has been applied to the detection of hGH in the sera of patients with high growth hormone dwarfism (Laron dwarfism). Substantial quantities of receptor-active hGH were found in the sera of all 7 patients studied. In one patient, arginine infusion elicited a prompt increase in both immunoactive and receptor-active hGH. These observations suggest that circulating hGH in Laron dwarfism is biologically active and support the concept that the disease may be caused by a generalized defect in hGH receptors.

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