Insulin In Human Plasma Research Articles

Circulating Sphingolipids, Insulin, HOMA-IR, and HOMA-B—The Strong Heart Family Study

High levels of fasting plasma insulin, reflecting insulin resistance, are an early event in the progression to type 2 diabetes. Experimental studies implicate ceramides in insulin resistance; however, whether circulating ceramides and related sphingolipids are associated with plasma insulin in humans is uncertain. We measured 15 ceramide and sphingomyelin species with different saturated fatty acids in baseline fasting plasma samples from the Strong Heart Family Study, a prospective cohort of American Indians. We examined associations of sphingolipids with plasma insulin, HOMA-IR and HOMA-B at baseline and follow-up (median 5 years later) using linear mixed models adjusted for family structure. Among 2086 participants without diabetes, higher levels of plasma ceramides carrying the fatty acids 16:0, 18:0, 20:0 or 22:0 were associated with higher plasma insulin and higher HOMA-IR at baseline and follow-up (Table). BMI modified associations of sphingomyelin species carrying 18:0, 20:0, 22:0 or 24:0 with insulin (p<0.003); higher sphingomyelin levels were associated with lower fasting insulin and HOMA indices only among those with normal BMI. Our study suggests lowering circulating ceramides might be a target in prediabetes, and targeting sphingomyelins should take into account BMI. Table. Plasma Ceramides, Insulin and HOMAIR.SpeciesFold change in geometric mean with 2 fold higher ceramide (95% CI), p-valueBaseline insulinFollow-up insulinBaseline HOMAIRFollow-up HOMAIRCer-16:01.13 (1.07-1.20) 1.9x10-51.10 (1.01-1.21) 0.031.15 (1.08-1.22) 4.7x10-61.11 (1.01-1.22) 0.03Cer-18:01.11 (1.06-1.15) 1.0x10-61.12 (1.06-1.19) 1.0x10-51.12 (1.08-1.17) 6.3x10-81.13 (1.06-1.20) 2.0x10-4Cer-20:01.12 (1.07-1.17) 1.5x10-71.10 (1.03-1.18) 0.0031.13 (1.08-1.18) 1.3x10-71.11 (1.04-1.19) 0.002Cer-22:01.13 (1.08-1.19) 3.9x10-71.12 (1.04-1.20) 0.0041.15 (1.09-1.21) 1.0x10-71.13 (1.04-1.22) 0.004Cer-24:01.(0.99-1.11) 0.081.09 (1.01-1.19) 0.041.(1.00-1.13) 0.041.11 (1.01-1.21) 0.03Covariates: age, sex, site, education, smoking, log(BMI), waist, physical activity. Disclosure R.N. Lemaitre: None. C. Yu: None. A. Hoofnagle: Research Support; Self; Waters Corporation. H. Nair: None. P.N. Jensen: None. A.M. Fretts: None. J.G. Umans: None. C. Sitlani: None. D. Siscovick: None. I. King: None. N. Sotoodehnia: None. B. McKnight: None.

Lixisenatide accelerates restoration of normoglycemia and improves human beta-cell function and survival in diabetic immunodeficient NOD-scid IL-2rg(null) RIP-DTR mice engrafted with human islets.

ObjectiveGlucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents, increases proliferation and survival of pancreatic beta cells. To investigate the effects on human beta cells, we used immunodeficient mice transplanted with human islets. The goal was to determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human islet function and survival in vivo.MethodsFive independent transplant studies were conducted with human islets from five individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with lixisenatide (50, 150, and 500 µg/kg) or vehicle. Islet function was determined by blood glucose, plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total beta- and alpha-cell number, percent proliferation, and levels of apoptosis.ResultsDiabetic mice transplanted with marginal human islet mass and treated with lixisenatide were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests, human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were significantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated mice. Nevertheless, in one experiment we found a significant twofold to threefold increase in human beta-cell numbers in lixisenatide-treated compared to vehicle-treated mice.ConclusionDiabetic human islet-engrafted immunodeficient mice treated with lixisenatide show improved restoration of normoglycemia, human plasma insulin, and glucose tolerance compared to vehicle-treated mice engrafted with the same donor islets. Because the proliferative capacity of human beta cells is limited, improved beta-cell survival coupled with enhanced beta-cell function following lixisenatide treatment may provide the greatest benefit for diabetic patients with reduced functional islet mass.

Rosalyn Sussman Yalow

Renowned medical physicist Rosalyn Sussman Yalow, a co-recipient of the 1977 Nobel Prize in Physiology or Medicine, died on 30 May 2011 in New York City.Rosalyn was born on 19 July 1921 in New York City. Although her parents were not able to attend high school, they passed down to Rosalyn their lifetime love of learning. Her fondness for chemistry was influenced by her high school chemistry teacher. In 1941 she became the first physics graduate of Hunter College, part of the City University of New York system and at that time a women’s college. Her love of physics was inspired by two of her professors there.In pursuing a graduate education, Rosalyn faced several obstacles: She was a woman in a man’s field, she was Jewish, and she spoke with the broad accents of New York City. With her usual determination, however, she was able to attend graduate school at the University of Illinois at Urbana-Champaign, where she started in 1941. Of the 400 people in the College of Engineering, she was the only woman. Maurice Goldhaber, who would later direct Brookhaven National Laboratory, served as her adviser. In 1943 she married physics graduate student Aaron Yalow, also a New Yorker and the son of a rabbi. Two years later Rosalyn received her PhD in nuclear physics—much of her research involved radioactivity. She received encouragement from Gertrude Goldhaber, a practicing physicist married to Maurice.After graduate school, Rosalyn took a job in New York at an ITT laboratory until it left the area a year later. She then returned to Hunter, this time to teach. Aaron, who by that time had joined Montefiore Hospital in the Bronx, introduced her to Edith Quimby, a trailblazer in medical physics. Quimby brought together Rosalyn and Gioacchino Failla, a pioneering biophysicist at Columbia University, who was very impressed with Rosalyn. Failla then convinced Bernard Roswit, chief of radiation therapy at the Bronx Veterans Administration Hospital, to hire her. She would spend her entire career in the VA hospital system.In 1950, while at the Bronx VA Hospital, she began her 22-year collaboration with internist Solomon Berson. Together they developed radioimmunoassay, a revolutionary technique that uses radiolabeled antibodies to measure antigens. They focused their research on the clinical diagnosis of thyroid diseases and the kinetics of iodine metabolism. The team developed an interest in serum proteins. At a time when highly purified insulin was readily available, Rosalyn and Berson soon determined that patients treated with animal insulins built up antibodies to them. They recognized that they had in their hands a means to measure circulating insulin. By 1959 they were able to put to practical use the measurement of plasma insulin in humans. Radioimmunoassay testing has now been largely replaced with fluorescent techniques; however, immunoassay is still used in routine blood tests.Berson died in 1972 and never saw the full recognition of his and Rosalyn’s work. In 1976 Rosalyn was presented with the Albert Lasker Basic Medical Research Award, considered by many to be a precursor to the Nobel Prize. The following year she was a co-recipient of the Nobel Prize in Physiology or Medicine. She was again recognized in 1988, this time with the National Medal of Science, for her groundbreaking work.Rosalyn and Aaron were deeply involved in the establishment of organizations dedicated to medical physics. In 1948 they helped found the Radiological and Medical Physics Society of New York, and in 1958 they became charter members of the American Association of Physicists in Medicine.Rosalyn took great pride in the many physicians and scientists that she and Berson mentored. Later in life, to ease the public’s fears about radiation exposure, she spoke in numerous US venues on the topic.Some 70 years after Rosalyn was the only woman in her graduate program, it is tantalizing to ask how things have changed. She would be the first person to start that discussion. She had made it her mission to inspire talented women in their pursuit of science and mathematics careers. Although Rosalyn would never classify herself as a feminist, many women scientists saw her as a role model, just as she viewed Marie Curie. Those of us to whom she has passed the torch have lost a champion and mentor. We will try to live up to her challenge.Rosalyn Sussman YalowAIP/ESVA, PHYSICS TODAY COLLECTIONPPT|High resolution© 2012 American Institute of Physics.

Determination of Insulin in Humans with Insulin-Dependent Diabetes Mellitus Patients by HPLC with Diode Array Detection

A simple and reliable high-performance liquid chromatographic method with diode array detection has been developed and validated for the determination of insulin in human plasma. A good chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 0.2M sodium sulfate (pH 2.4), 25:75 (v/v). Its flow rate was 1.2 mL/min. Calibration curve was linear within the concentration range of 0.15-25 µg/mL. Intra-day and inter-day relative standard deviations for insulin in human plasma were less than 6.3 and 8.5%, respectively. The limits of detection and quantification of insulin were 0.10 and 0.15 µg/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of insulin in eight insulin-dependent diabetes mellitus patients after subcutaneous injection of 25 IU of Actrapid HM.

Insulin Resistance and Alzheimers Disease Pathogenesis: Potential Mechanisms and Implications for Treatment

Insulin modulates cognition and other aspects of normal brain function. Insulin resistance is characterized by chronic peripheral insulin elevations, and it is accompanied by reduced brain insulin levels and insulin activity. Obesity, type 2 diabetes mellitus and hypertension are strongly associated with insulin resistance. In addition, insulin resistance increases the risk of age-related memory impairment and Alzheimer's disease. Possible mechanisms through which these risks are increased include the effects of peripheral hyperinsulinemia on memory, CNS inflammation, and regulation of the beta-amyloid peptide. We have shown that raising plasma insulin in humans to levels that characterize patients with insulin resistance increases the levels of Abeta and inflammatory agents in brain. These convergent effects may impair memory and induce AD pathology. Therapeutic strategies focused on preventing or correcting insulin abnormalities may thus benefit a subset of adults with age-related memory impairment and AD.

A Luminescent Oxygen Channeling Immunoassay for the Determination of Insulin in Human Plasma

The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 microL. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R(2) = 0.98). This method is convenient and represents an alternative to ELISA.

Gene Transfer of Constitutively Active Akt Markedly Improves Human Islet Transplant Outcomes in Diabetic Severe Combined Immunodeficient Mice

Akt is an important intracellular mediator of beta-cell growth and survival in rodents. However, whether constitutive activation of Akt in human beta-cells enhances the survival and function of transplanted islets is unknown. In the current study, we examined the efficacy of constitutive activation of Akt in improving human islet transplant outcomes using a marginal mass model in diabetic severe combined immunodeficient (SCID) mice. Human islets transduced with adenoviruses encoding constitutively active Akt1 (Adv-CA-Akt) displayed increased total and phosphorylated Akt and Akt kinase activity compared with control islets. Expression of CA-Akt in human islets induced a significant increase in beta-cell replication and a significant decrease in beta-cell death induced by serum and glucose deprivation or chronic hyperglycemia. Two control groups of islets (1,500 uninfected or adenovirus LacZ [Adv-LacZ]-transduced human islet equivalents [IEQs]) transplanted under the kidney capsule of streptozotocin-induced diabetic SCID mice were insufficient to correct hyperglycemia. Importantly and in marked contrast to these controls, 1,500 Adv-CA-Akt-transduced IEQs were capable of restoring euglycemia in diabetic SCID mice. Moreover, blood glucose normalization persisted for at least 6 months. Human plasma insulin at day 54 after transplant was 10-fold higher in Adv-CA-Akt islet recipients (2.4 +/- 0.4 ng/ml) compared with those receiving Adv-LacZ islets (0.25 +/- 0.08 ng/ml) (P < 0.05). In summary, expression of CA-Akt in human islets improves islet transplant outcomes in a subcapsular renal graft model in SCID mice. Akt is an attractive target for future strategies aimed at reducing the number of islets required for successful islet transplantation in humans.

Qualitative Determination of Synthetic Analogues of Insulin in Human Plasma by Immunoaffinity Purification and Liquid Chromatography−Tandem Mass Spectrometry for Doping Control Purposes

Synthetic insulins such as Humalog Lispro, Novolog Aspart, or Lantus Glargine, are commonly employed for the treatment of insulin-dependent diabetes mellitus owing to convenient handling and fast or prolonged bioavailability. However, the misuse of insulin in sports has been reported often, and the international doping control system requires a reliable and robust assay to determine the presence or absence of related drugs prohibited by the World Anti-Doping Agency. Qualitative evidence of administered substances, which is preferably obtained by mass spectrometry, is of utmost importance. Plasma specimens of 2 mL were fortified with three synthetic insulin analogues and purified by immunoaffinity chromatography, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This top-down sequencing-based assay allows the assignment of individual fragment ions, in particular, of those comprising modifications that are originating from C-termini of B-chains. Recoveries of synthetic insulins from plasma aliquots ranged from 91 to 98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes.

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans.

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1c) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M(r) of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M(r) 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to approximately 9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 micro g x kg(-1) x min(-1), followed by 2 h at 83.0 micro g x kg(-1) x min(-1); corresponding to 0.4 and 2.0 mU x kg(-1) x min(-1)). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P < 0.01) and approximately 70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes.

Hormonal FM: what's the frequency?

Recently, Peter Bergsten and co-workers measured slow oscillations (∼0.2 min−1) in the oxygen tension (pO2) and cytosolic calcium concentration {[Ca2+]} that correlated with the frequency of pulsatile insulin secretion into the portal vein [1xPrimary in vivo oscillations of metabolism in the pancreas. Bergsten, P. et al. Diabetes. 2002; 51: 699–703Crossref | PubMedSee all References[1]. Given the roles of [Ca2+] and pO2 in stimulating insulin secretion, these data provide strong evidence that secretion from individual islets, located at various distances from the portal vein, is coordinated throughout the pancreas. The frequency signature can only be maintained if the hormone moves through the pancreas quickly; the authors estimated the passage rate of insulin by measuring that of glucose. Although these studies targeted the portal vein, others have reported oscillations of approximately the same frequency in human plasma insulin [2xCyclic oscillations of basal plasma glucose and insulin concentrations in human beings. Lang, D.A. et al. N. Engl. J. Med. 1979; 301: 1023–1027Crossref | PubMedSee all References[2]. Similar to other blood-borne signaling molecules, the pulsatile nature of plasma insulin is important for promotion of the hormone's action.Bergsten's results raise many questions regarding the mechanisms, not only for coupling the fluctuating levels of cytosolic signals to the exocytosis of insulin, but also for coordinating secretion from individual islets. Citing the lack of synchronous electrical oscillations between islets within the same pancreas, the authors suggest that the rapid passage of glucose through the pancreas implies that coordination might be achieved by a blood-borne agent. The relationships between cellular carbohydrate metabolism, cytosolic [Ca2+] and secretion of insulin, are well studied. However, that these parameters also oscillate in response to glucose in cultured cells is less well appreciated. These in vivo findings suggest that the pulsatile nature of signaling molecules – be they blood-borne compounds (e.g. insulin and non-esterified fatty acids) or intracellular signals {e.g. [Ca2+] and ADP/ATP} – should be considered when describing the machinery that uses them to regulate fuel metabolism. The relevance of this work to the highly popular metabolic clamp approach, in which the blood and/or tissue content of select molecules is held constant, remains to be determined. Even so, the metabolic clamp has provided significant advances in our understanding of in vivo regulatory mechanisms.Although not discussed by the authors, the in vivo oscillation of insulin levels raises the possibility that some portion of the blood-borne insulin signal is carried by the frequency of the pulses, not simply by the level of insulin. The ability of extra-pancreatic tissues to receive this putative frequency-modulated (FM) signal has not been sufficiently investigated. Even so, oscillations of fructose-1,6-bisphosphate (F16P2) and ADP/ATP have been reported in skeletal muscle extracts [3xFructose-2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts. Tornheim, K. J. Biol. Chem. 1988; 263: 2619–2624PubMedSee all References[3]. If such an FM signaling system is actually functioning in vivo, then it might constitute another regulatory mechanism for coordinating whole-body fuel metabolism.

A radioimmunoassay method for determination of fish plasma insulin.

The present study was undertaken to clarify whether the radioimmunoassay for the determination of plasma insulin in humans is applicable to quantify that of the fishes, and to improve the assay method. In experiments in which 125I-skipjack insulin was used as a tracer, human insulin did not react to the anti-skipjack insulin serum, whereas skipjack insulin reacted very strongly. Red sea bream and yellowtail insulins showed a similar reaction to skipjack insulin. It was also found that the fish plasma can be employed as the blood plasma which is added into the assay system of standard skipjack insulin. On the basis of these findings, a modified method was presented to determine the plasma insulin of fishes. Furthermore, when this modified method was used, the B/T ratio of standard skipjack insulin exhibited little deviation at various levels, and also high recovery was obtained for red sea bream insulin. These results indicate that this method can be applied to determine the plasma insulin of fishes.

Direct Radioimmunoassay of Proinsulin and Insulin in Human Plasma by Chromatographic Technique

Specific method for direct radioimmunoassay of IRP and IRI separately in human plasma has been described. The method is used for extraction of total insulin and separation of IRP from IRI by paper chromatography to be assayed separately. The separation of the two components are indentified and confirmed by column chromatography, paper chromatography and U.V. spectral analysis in comparison with the standard compounds. 134 plasma samples of different cases were, investigated for determination of IRI, IRP and IRT, of which 39 normals, 16 normal obes, 21 juvinil diabetes, 18 adult oncet diabetes, 10 recent adult diabetes, 12 hypothroid and 18 bilharzial hepatosplenomegaly to evaluate the levels of the test in comparison with blood sugar concentration.

Inhibition by somatostatin of insulin release from isolated pancreatic islets

Since the finding of somatostatin, the hypothalamic inhibitor of pituitary growth hormone, many experimental data showing the inhibiting activity of somatostatin on the secretion of other hormones have been presented [ 11. As for the insulin secretion, somatostatin was reported to suppress a glucoseinduced rise of plasma insulin in humans [2] and to inhibit insulin secretion by perfused pancreases [2-41. However, concerning its direct effect on insulin release from the pancreatic islet of Langerhans there have so far been two controversial results; Although Efendic et al. [3] failed to demonstrate the inhibitory effect of somatostatin on insulin release from the isolated islet, Oliver and Wagle [5] have quite recently reported that the inhibition of insulin release from the islet was observed in experiments with incubation media containing somatostatin concentrations ranging from 0.2 to 100 pglml. Recently, we have isolated rat pancreatic islets under mild conditions of collagenase treatment, and studied the effect of somatostatin on glucose-induced insulin release by the islet. The purpose of present paper is to show that somatostatin, in such a low concentration as 10 &ml, did inhibit insulin release from the islet and that the somatostatin inhibition of insulin release was completely reversed by increasing Ca” concentration in the incubation medium.

Measurement of and Correction for Incubation Damage in Radioimmunoassay

Abstract. The role of incubation damage in the radioimmunoassay of human plasma insulin, human plasma growth hormone and rat plasma growth hormone was studied. Damage was measured as the fraction of labelled material which migrated from the origin in wick paper chromatography of samples without antibody. The experiments demonstrated, that recovery of added hormone differed increasingly from 100% as incubation damage increased. Recoveries were found to be too high using the double antibody technique, and too low using chromatographic technique. However, satisfactory recoveries could be obtained in chromatographic assay after correction for the experimentally determined incubation damage using the general formula: % Bound=100 × (B‐KD)/(100‐KD) where B is the apparent antibody‐bound fraction, D the damage fraction, both expressed as percent of the total radioactivity. K is determined experimentally.

Measurement of and correction for incubation damage in radioimmunoassay.

Abstract. The role of incubation damage in the radioimmunoassay of human plasma insulin, human plasma growth hormone and rat plasma growth hormone was studied. Damage was measured as the fraction of labelled material which migrated from the origin in wick paper chromatography of samples without antibody. The experiments demonstrated, that recovery of added hormone differed increasingly from 100% as incubation damage increased. Recoveries were found to be too high using the double antibody technique, and too low using chromatographic technique. However, satisfactory recoveries could be obtained in chromatographic assay after correction for the experimentally determined incubation damage using the general formula: % Bound=100×(B‐KD)/ (100‐KD) where B is the apparent antibody‐bound fraction, D the damage fraction, both expressed as percent of the total radioactivity. K is determined experimentally.

A radioimmunoassay for plasma insulin using zirconyl phosphate gel

A procedure for the detection of immunoreactive insulin in human plasma is described. The procedure is a rapid radioimmunoassay (3 hr) which uses zirconyl phosphate gel (Z-gel) at a pH of 6.25 to adsorb antibody-bound insulin while leaving free insulin in solution. Thirty-two samples of human plasma which had been assayed for immunoreactive insulin using a commercially available assay (Pharmacia Fine Chemicals, Piscataway, NJ) were also assayed on a blind basis for immunoreactive insulin using the Z-gel technique. A statistical comparison of the data obtained by the two methods yielded a highly significant ( P < 0.01) coefficient of correlation (0.998). A known amount of bovine insulin was added to each of these plasma samples, and the Z-gel assay repeated. The mean percentage recovery of added insulin was 100.4 ± 17.0 (SD). In addition, the insulin response of two healthy individuals to the ingestion of 100 g of glucose was determined using the Z-gel assay. The insulin response curves were similar to those described by other investigators.

A method for the determination of plasma insulin antibodies and its application in normal and diabetic subjects.

A practical, sensitive and specific method for the assay of antibodies to insulin in human plasma has been developed. This method involves competition for insulin antibody between a constant, trace amount of I-125-insulin and increasing concentrations of unlabeled insulin standard. The insulin standard is prepared from the soluble form of therapeutic insulin that the patient has been receiving. The quantity of antibody is calculated arbitrarily from the amount of unlabeled insulin required to displace 25 per cent of bound I-125-insulin from the antibody. The result is expressed as microunits of insulin antibody per milliliter of plasma. Since this is not the absolute amount of antibody present but does indicate a relative, quantitative immunologic response of each patient to the antigen, insulin, the result is referred to as an antibody index. The method has a precision of 20 per cent and sensitivity of 1 μU./ml. The coefficient of variation is significantly less at higher antibody indices: 8 per cent in the 1,001 to 80,000 range and 22 per cent in the 0.08 to 40 μU./ml. range. Therapeutic subcutaneous doses of Lente or Sulphated insulin do not interfere with the antibody assay, whereas the interference from Regular insulin continues for several hours. The antibody index declines after withdrawal of insulin therapy and has a half-life of approximately eighteen days. There is no diurnal variation in antibody index. Three newborn infants of insulin-treated diabetic mothers were found to have insulin antibodies.

Significance of human plasma insulin Sephadex fractions.

Fifty-nine samples taken from lean and obese healthy subjects and from patients with diabetes mellitus, myotonic dystrophy, and islet cell adenoma after the administration of oral or intravenous glucose or other insulin secretogogues were fractionated on Sephadex G-50 columns. Eluates corresponding to zones of elutions of porcine pro insulin (“big insulin”) and Regular insulin were assayed for insulin by radioimmunoassay using crystalline human insulin as standard and porcine insulin-I-125 as tracer. In four samples from one patient with islet cell adenoma the big insulin fraction composed 24 to 55 per cent of the apparent insulinimmunoreactivity but in no other case did big insulin exceed 20 per cent of the total and in fifty-one of the fifty-five samples other than from the patient with insulinoma, big insulin contributed less than 15 per cent to the total apparent immunoreactive plasma insulin.

Use of immunosorbent in the preparation of an “antigen-free” plasma. An adequate diluent for standards in radioimmunoassay

An immunosorbent was produced by coupling insulin antibodies to Sephadex. By means of this, endogenous insulin in human plasma could be absorbed, giving an insulin-free plasma, which was unaltered in other respects. Radioimmunoassay of insulin with standards containing or not containing such insulin-free plasma showed that the antigen-antibody reaction was influenced by the presence of plasma. This implies that for the measurement of an antigen by radioimmunoassay such an “antigen-free” blank should be included in the standards to make the conditions for the reactions as well as the degradation of the labelled antigen identical in standards and unknown samples.

Experience with the immunoprecipitation technique of insulin assay with reference to sensitivity, precision and specificity

Experience with the immuno-assay system of Hales and Randle 4 is given. With insulin anti-sera provided by Burroughs Wellcome, and labelled insulin from the Radiochemical Centre, a satisfactory assay system for human plasma insulin was possible. The assay has been shown to be precise, sensitive and specific. Care with the minor points of technique is stressed.