A B S T R A C T A preparative scheme has been developed for the purification of a trace protein in human serum exhibiting nonsuppressible insulin-like activity (NSILA). This scheme consisted of (a) adsorption chromatography of serum utilizing the sulfonic acid polystyrene resin, Dowex 50, at pH 6.8; (b) G-200 gel filtration at pH 8.9; and (c) acrylamide gel electrophoresis in a discontinuous preparative system. Throughout all procedures, NSILA fractionated as a single molecular species approximating 90.000 mol wt. The purified protein exhibited a single band by disk gel electrophoresis, an isoelectric pH approximating 6.2, doublet bands of 90,000 and 92,000 mol wt by analytical sodium dodecyl sulfate gel electrophoresis, and a biologic specific activity approximating 50 mU/mg. Serum somatomedin (sulfation factor) activity did not fractionate with NSILA in this scheme, and partially purified NSILA did not stimulate radiosulfate uptake into hypophysectomized rat costal cartilage. This protein appears to represent the major constituent of serum NSILA: its purification and partial characterization provides the first step towards elucidation of its metabolic role.

This study was designed to focus on the genetic control of tolbutamide disposition in humans and to provide insight into the potential for high accrued blood levels in individuals receiving fixed dosage regimens. Tolbutamide was administered intravenously to 42 nondiabetic subjects, eight of their relatives, and to five sets of twins. A ninefold variation in the rate of tolbutamide disappearance from plasma (Kd) was found. This variation was characterized by a trimodal frequency distribution, suggestive of monogenic inheritance and consistent with pedigree analysis, indicating autosomal transmission of rapid and slow inactivation of tolbutamide. A heritability value of 0.995 for Kd indicated little influence of environmental factors on variation of this rate. Interindividual differences in the binding of 35S-tolbutamide to serum proteins were also assessed. No correlation was found between tolbutamide serum protein binding affinity and Kd. Analysis of the metabolites of tolbutamide in urine samples provided evidence for the microsomal oxidation of the drug to hydroxytolbutamide as the primary site of genetic control. In conclusion, this study provides evidence for monogenic control of tolbutamide metabolism in man. The results suggest that fixed dosage regimens of this drug, as were prescribed in the controversial University Group Diabetes Program study, might lead to higher accrued blood levels in slow inactivators.

The storage stability of 3-hydroxybutyrate in whole blood, serum, and plasma was evaluated. The levels of 3-hydroxybutyrate were measured using an enzymatic rate reaction of the specific 3-hydroxybutyrate dehydrogenase with the NAD-NADH coupled reaction. 3-Hydroxybutyrate was found to be a stable analyte in whole blood, plasma, and serum. The long-term stability of beta-hydroxybutyrate allows ample time for separation of blood components and offers storage of samples to meet quality control needs as well as the possibility of mailing specimens. Further studies indicate that NaF plasma, heparin plasma, and serum are the preferred specimens, because both EDTA and oxalate showed the most significant interference with the determination of 3-hydroxybutyrate.

Because ILA was encountered in TCA-ethanol extracts of plasma dialyzed in boiled Visking casing which interfered with measurement of the "synalbumin insulin antagonist" on isolated muscle, a systematic study of the influence of the casing on these factors was undertaken. Plasma from healthy humans was extracted with TCA-ethanol and dialyzed in nonboiled and boiled Visking casing. The extract ("albumin") was tested by measurement of glucose uptake in rat hemidiaphragms, without and with insulin (1,000 /iU./ml.).. Significant amounts of both ILA and anti-insulin activity were detected in both "albumins" at 3 to 5 gm. per cent; however, more ILA and less insulin inhibition was found in "albumin" dialyzed in boiled casing. The magnitude of ILA could not be accounted for by immunoreactive insulin levels in the "albumin," and the ILA was not suppressed by insulin-specific antiserum (NSILA).From dose-response curves measuring glucose uptake and glycogen synthesis, more NSILA was detected in the "albumin" from boiled casing; however, at 3 to 5 gm. per cent concentration, "albumin" dialyzed in either casing caused inhibition of NSILA. Because partially purified NSILA was found to be retained equally by either boiled or nonboiled casing, an antagonist in Yisking casing was sought. A substance from Visking casing with small molecular weight was found to cause insulin inhibition. When nonantagonistic crystalline albumin was dialyzed in nonboiled casing, marked antagonism to both crystalline insulin and partially purified NSILA was demonstrated.It is concluded that an artifactual casing antagonist to both NSILA and insulin is bound to albumin and may contribute to the measurement of the "synalbumin insulin antagonist.

AB STRACT Studies were undertaken in an attempt to clarify the apparent heterogeneous nature of human serum insulin-like activity. Methods of preparative zone electrophoresis on Pevikon, acid-ethanol extraction of trichloroacetic acid serum protein precipitates, adsorption chromatography on DEAE-cellulose and Dowex 50, gel filtration chromatography, and insulin antiserum immunoreactivity were used. The results establish the presence of a substance in serum with in vitro biological properties similar to insuln but with different physicochemical properties. The major portion of serum ILA measured by bioassay techniques can be attributed to the effects of this substance. Whereas the in vitro biological effects of this substance on muscle and adipose cells were similar to those of crystalline insulin, the substance is distinguished from insulin by: (1) the failure of insulin antiserum to inhibit its in vitro biological effect; (2) a slower electrophoretic mobility (in the gamma-beta globulin zone); and (3) a larger molecular weight, between 40,000 and 50,-000 in these studies. It is similar to insulin since both are soluble in acid-ethanol. The results furThis work was presented in part as an abstract at the 26th Annual Meeting of The American Diabetes Association.