Insulin In Urine Research Articles

Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS.

The increasing importance to determine bioactive peptide hormones such as insulin, its synthetic analogs, and C-peptide in urine samples represents an analytical challenge. The physiological concentrations of insulin in urine are commonly found at sub-ng/mL levels and thus represent a complex analytical task. C-peptide concentrations, on the other hand, tend to be in the moderate ng/mL range and are hence much easier to determine. Insulin and C-peptide are important in the diagnostics and management of metabolic disorders such as diabetes mellitus and are also particularly relevant target analytes in professional sports and forensics. All insulins are classified on the World Anti-Doping Agency’s (WADA) list of prohibited substances and methods in sports with a minimum required performance level (MRPL) of 50 pg/mL. Until now, methods combining immunoextraction and subsequent mass spectrometric detection have mostly been used for this purpose. With the method developed here, sample preparation has been simplified considerably and does not require an antibody-based sample purification. This was achieved by a sophisticated mixed-mode solid-phase extraction and subsequent separation with liquid chromatography coupled to high-resolution mass spectrometry. Included target insulins were human, lispro, glulisine, aspart, glargine metabolite, degludec, and additionally, human C-peptide. The method was validated for the synthetic insulin analogs considering WADA requirements including specificity, limit of detection (10–25 pg/mL), limit of identification, recovery (25–100%), robustness, carry over (<2%), and matrix effects. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely, [[2H10] LeuB6, B11, B15, B17]-insulin and [[13C6] Leu26, 30] C-peptide. Finally, the method was applied to samples from patients with diabetes mellitus treated with synthetic insulins.

Towards personalised molecular feedback for weight loss

BackgroundNumerous diets, apps and websites help guide and monitor dietary behaviour with the goal of losing weight, yet dieting success is highly dependent on personal preferences and circumstances. To enable a more quantitative approach to dieting, we developed an integrated platform that allows tracking of life-style information alongside molecular biofeedback measurements (lactate and insulin).MethodsTo facilitate weight loss, participants (≥18 years) omitted one main meal from the usual three-meal routine. Daily caloric intake was restricted to ~1200KCal with one optional snack ≤250KCal. A mobile health platform (personalhealth.warwick.ac.uk) was developed and used to maintain diaries of food intake, weight, urine collection and volume. A survey was conducted to understand participants’ willingness to collect samples, motivation for taking part in the study and reasons for dropout.ResultsMeal skipping resulted in weight loss after a 24 h period in contrast to 3-meal control days regardless of the meal that was skipped, breakfast, lunch or dinner (p < 0.001). Common reasons for engagement were interest in losing weight and personal metabolic profile. Total insulin and lactate values varied significantly between healthy and obese individuals at p = 0.01 and 0.05 respectively.ConclusionIn a proof of concept study with a meal-skipping diet, we show that insulin and lactate values in urine correlate with weight loss, making these molecules potential candidates for quantitative feedback on food intake behaviour to people dieting.

Recent advances in the determination of insulins from biological fluids.

The qualitative and quantitative determination of insulin and its related substances (e. g., C-peptide) is of great importance in many different areas of analytical chemistry. In particular, due to the steadily increasing prevalence of metabolic disorders such as diabetes mellitus, an adequate control of the circulating amount of insulin is desirable. In addition, also in forensics and doping control analysis, the determination of insulin in blood, urine or other biological matrices plays a major role. However, in order to establish general reference values for insulin and C-peptide for diabetology, the comparability of measured concentrations is indispensable. This has not yet been fully implemented, although enormous progress has been made in recent years, and the search for a "gold standard" method is still ongoing. In addition to established ligand-binding assays, an increasing number of mass-spectrometric methods have been developed and employed as the to-date available systems (for example, high-resolution/high accuracy mass spectrometers) provide the sensitivity required to determine analyte concentrations in the sub-ng/mL (sub-100pmol/L) level. Meanwhile, also high-throughput measurements have been realized to meet the requirement of testing a high number of samples in a short period of time. Further developments aim at enabling the online measurement of insulin in the blood with the help of an insulin sensor and, in the following, in addition to a brief review, today's state of the art testing developments are summarized.

A supported liquid membrane for microextraction of insulin, and its determination with a pencil graphite electrode modified with RuO2-graphene oxide

A new method for the microextraction and electrochemical determination of insulin is presented based on the following steps: (1) extraction of insulin from an aqueous phase (the donor phase) at pH 8.0 into a sodium dodecyl sulfate (SDS) supported liquid membrane phase to form an insulin-dodecyl sulfate complex, (2) re-extraction of insulin into a small volume of an acceptor phase at pH 3.0 (leading to preconcentration), and (3) detection by differential pulse voltammetry (DPV) using a new kind of electrode. The effects of the pH values of the donor and acceptor phases, SDS concentration, percentage of organic solvent, extraction time and ionic strength on the microextraction were optimized. A pencil graphite electrode modified with graphene oxide and ruthenium dioxide was used as an electrochemical sensor. The DPV responses to the concentration of insulin was linear in the range 0.0008–0.020 and 0.02–1.00 μmol L−1, with a detection limit of 24 pmol L−1. The technique was successfully applied to the determination of insulin in human urine and plasma.

Serum insulin and C-peptide levels as markers of pre-eclampsia in pregnant women

Objectives This study aimed to detect the relation between fasting C-peptide level and serum insulin level in pre-eclamptic pregnant women. Background Pre-eclampsia is a systemic disease that is characterized by increased vascular resistance, endothelial dysfunction, proteinuria, coagulopathy, and hypertension. Similarities in certain biochemical variables between pre-eclampsia and the insulin resistance syndrome imply a possible link between insulin resistance and pre-eclampsia. Patients and methods A total of 60 pregnant women were included in this study after the approval of the ethical committee of the Alexandria University (where the study was conducted in); then, they were divided into two groups: 30 normal pregnant women as controls and another 30 pregnant women with the complication of pre-eclampsia served the study group. This group was subdivided into 10 patients with severe pre-eclampsia and another 20 with mild pre-eclampsia. All the patients were subjected to the oral glucose tolerance test (OGTT), assessment of fasting serum insulin and fasting C-peptide, and urine for dipstick. Results A statistically significant difference was found between the two groups in the mean arterial blood pressure, systolic blood pressure, and diastolic blood pressure. There was a statistically significant difference between the two groups in BMI as increased BMI was associated with pre-eclampsia. There were statistically significant differences between the two groups in serum C-peptide, serum insulin, and protein in urine. Conclusion Serum C-peptide and serum insulin levels were significantly lower in women with severe pre-eclampsia than in women with mild pre-eclampsia and normotensive pregnant women, with weak associations to pre-eclampsia. BMI was significantly higher in women with severe and mild pre-eclampsia than in normotensive pregnant women, with strong associations to pre-eclampsia.

Doping control analysis of insulin and its analogues in equine urine by liquid chromatography–tandem mass spectrometry

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography–tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05 ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4 h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.

Synthesis, characterization and application of modified Pd nanoparticles as preconcentration probes for selective enrichment/analysis of proteins via hydrophobic interactions from real‐world samples using nanoparticle‐liquid‐liquid microextraction coupled to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

AbstractWe introduce a novel preconcentrating technique by using surface modification of palladium nanoparticles (Pd‐NPs) with octadecane thiol (ODT) prepared in toluene for selective and sensitive extraction of proteins (insulin, ubiquitin, lysozyme) from a variety of real‐world samples including pancreas, mushroom, soybean and milk using nanoparticle‐liquid‐liquid microextraction (NP‐LLME) coupled to matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐MS). The limit of detection (LOD) values obtained for gramicidin D and insulin in water and urine are between 17–37 nM (17–37 fmol) (with RSDs ranging from 5.3–7.2%) which are 10–20‐fold enhancement in detection sensitivity compared with conventional MALDI‐MS. The optimal sample pH for highest extraction efficiency of insulin, ubiquitin and lysozyme from biological samples was observed at sample pH ∼ pI which could be due to the enhancement of hydrophobic interactions between proteins with the hydrophobic ligands of Pd‐ODT NPs. In addition, we also found that with the addition of 1 M NaCl, signals could be significantly enhanced by using the current approach. It is an efficient, straightforward, sensitive and selective nanoprobe which can be widely applied for separation, enrichment and preconcentration of peptides or proteins from complex biological samples in proteome research. Copyright © 2010 John Wiley &amp; Sons, Ltd.

Invalidation by extreme assay artefacts of immunoassayable urinary insulin as a parameter of pancreatic allograft function

There are several reasons to believe that declines in pancreatic beta cell function should presage clinical rejection of pancreatic allografts; furthermore, assessments of urinary insulin might be more informative than plasma determinations to assess pancrcaticocystostomy function. However, the previous literature on urinary insulin is inconsistent. We examined endogenous insulin values, and recovery of exogenously‐added insulin in urine from control subjects and patients, before and after ultrafiltration to remove proteolytic enzymes derived from the pancreas. In patients with a pancreatic graft, major artefacts in the radioimmunoassay were found (attributable to degradation of tracer and/or antibody, presumably by proteases) which could not be obviated by ultrafiltration or other approaches. These included spuriously high endogenous “insulin” levels, and virtually total inability to recover authentic insulin added to PxTx (pancreas transplant) urine. These findings raise significant doubts about the validity of most previous studies on urinary insulin (or other markers for pancreatic rejection) based on the use of immunoassay measurements.

Effect of acute renal failure on insulin disposition in the isolated perfused rat kidney.

The renal disposition of insulin in acute renal failure has not been evaluated. We used the isolated perfused rat kidney to test the hypothesis that acute renal failure (ARF) decreases renal insulin clearance. We used warm ischemia for 45 min, uranyl nitrate 5 mg/kg, ureter ligation, and nonfiltering kidneys as methods of inducing ARF. Comparisons were made with normal control kidneys. The concentrations of insulin in perfusate and urine was determined by radioimmunoassay. Acute renal failure caused significant reductions in glomerular filtration rate, sodium and potassium reabsorption, and an increased urine pH. Warm ischemia and uranyl nitrate toxicity caused a 50% decrease in the renal clearance of insulin. Nonfiltering kidneys cleared insulin at a rate 90% decreased from controls. Ureteral ligation caused a 32% decrease in insulin clearance. Filtration was necessary for insulin to be cleared from perfusate. We conclude that ARF decreased renal insulin clearance through a decrease in insulin uptake from both the tubular lumen and peritubular surface.

The excretion of insulin in urine

The concentration of biologically active insulin was measured by the isolated fat cell method in serum and urine from healthy subjects and compared with the concentration of immunoreactive insulin. In both urine and serum the values obtained by the two methods correlated closely; In addition, there was a close correlation between the concentration of biologically active and immunoreactive insulin in urine from maturity onset diabetics. Therefore, conclusions on the excretion in the urine and the urinary clearance of insulin, which are based on measurements of immunoreactive insulin, are also valid for biologically active insulin.

Studies in the renal handling of insulin in juvenile diabetics.

A technique is described for the accurate radioimmunoassay of insulin in serum and urine. This method was applied to study of renal clearance and excretion of endogenous and exogenous insulin in untreated juvenile diabetics and healthy young adults. There was good agreement between our results for normal adults and previously reported values. In six non-obese juvenile diabetics, urinary insulin clearance values, both basal (fasting) and following glucose loading (entire range 0.03 ml/min to 1.23 ml/min) were similar to those obtained for the adults (entire range 0.17 ml/min to 2.35 ml/min). The basal urinary excretion in these diabetics was generally of the same order of magnitude as that in the normals. The clearance of exogenous insulin, administered for the first time, was also of the same order as that for endogenous insulin. Markedly elevated urinary clearance and excretion of insulin during fasting and non-fasting states was demonstrated in four non-obese juvenile diabetics with no clinical evidence of abnormal proteinuria, though they demonstrated slight to mild clinical dehydration and acidosis compared with the other diabetics studied. Clearance and excretion of exogenous insulin was similarly elevated. This finding could reflect renal tubular dysfunction in these diabetics, and this dysfunction could relate to even the mild degree of dehydration and acidosis found in this study. Endogenous and exogenous insulin clearance in an obese diabetic child was similar to that for the control group.

The excretion of proinsulin and insulin in urine

Proinsulin-like components (PLC) and insulin have been measured in 24 hr urine samples from 8 healthy subjects. The mean excretion of PLC was 45.8 ng and that of insulin 314 ng; the PLC: insulin ratio was 0.14. Urinary PLC was increased 3.5 fold in a patient with a pancreatic islet cell tumor and the PLC: insulin ratio was 0.35. The urinary PLC: insulin ratio is lower than that of serum, presumably because of the relatively lower urinary clearance of the larger molecular weight PLC.

Insulin and the kidney.

Changes in renal function and structure are frequently observed in patients with diabetes mellitus. In the early phases of the disease, alterations in glomerular filtration rate, renal plasma flow, glomerular permeability and tubular capacity for glucose reabsorption occur. In the late stages of juvenile onset diabetes, renal failure is a common cause of death. For this reason, increasing attention is being paid to the possibility of long-term dialysis and renal transplantation in these patients. The kidneys play an important role in regulating insulin metabolism. The renal arteriovenous difference is approximately 30-45% and a linear relationship exists between the arterial insulin level and the renal arteriovenous concentration difference. The renal extraction of insulin is 200 ml/min in man, and it is estimated that 6-8 U are removed and degraded by the kidney in 24 h. The quantity of insulin in urine is small. However, its clearance is relatively constant over a wide range of serum concentrations and is 0.15-0.5 ml/min. The mean basal insulin excretion is 3.6 muU/mg creatinine, and a fourfold rise occurs following a glucose load. The urinary insulin values in neonates, children and patients with diabetes and renal failure are reviewed. In diabetic patients, progressive renal disease is accompanied by decreasing insulin requirements. In contrast, nondiabetic patients who develop renal failure frequently show abnormalities in carbohydrate metabolism, the commonest of which is a pseudodiabetic state.

Hypoglycemia and excretion of insulin in urine in hemolytic disease of the newborn.

Extract: Blood glucose levels and rates of excretion of insulin in urine were measured in 31 infants with erythroblastosis. Twenty-two of these infants had determinations of insulin excretion in urine performed during the first 24 h of life. These infants can be divided into two groups. Group 1 were infants of greater than 36 weeks gestation. When using hemoglobin as the index of severity of the disease, they can be subdivided into two groups. Group 1A (six infants with a cord hemoglobin value of greater than 13 g/100 ml) had a mean excretion ratio of insulin/creatinine in urine of 6.9 μU/mg compared with a mean of 5.4 μU/mg for the control group (P = 0.5). Group 1 B (six infants with a cord hemoglobin value of less than 13 g/100 ml) had a mean value for insulin in the urine of 47.0 μU/mg, compared with a mean value of 5.4 μU/mg for the control group. Group 2, composed of 10 infants of less than 36 weeks gestation, had a mean value for insulin in urine of 129.7 μU/mg, compared with a mean of 21.9 μU/mg for the control group (P < 0.01). Excretion of insulin was inversely related to blood glucose. Seven infants were hypoglycemic (blood glucose levels of less than 20 mg/100 ml) and all had elevated insulin levels in urine.Speculation: In erythroblastotic infants, simultaneous occurrence of islet cell hyperplasia, hypoglycemia, and hyperinsulinuria is indicative of increased insulin production. The cause of the islet cell hyperplasia is not apparent. The placenta is commonly hyperplastic, and hence, its ability to destroy insulin may be enhanced, thus causing islet cell hyperplasia. At birth, separation of the infant from the placenta leads to an abrupt decrease in the infant's rate of insulin destruction, and thus, to temporary over-production of insulin. This hypothesis is in keeping with the observed early hypoglycemia and the normal birth weight for gestational age.

Increase of Urinary Insulin Excretion Following Probenecid Administration in Man

Oral administration of probenecid augmented urinary clearance of, trichloroacetic acid precipitable 131I-insulin in six men who were injected with tracer dose of 131I-insulin intravenously. The increments were 16 to 423 % above the control value (P<0.05). In other eight men, endogenous insulin in urine and plasma was measured by radioimmunoassay method. Apparently increased responses in urinary insulin excretion and urinary insulin clearance were observed after probenecid administration. The percentage increases of the insulin excretion and the insulin clearance beyond the control values were 2 to 200 (P<0.01) and 7 to 200 (P<0.01), respectively.It was suggested that insulin was reabsorbed at the proximal convoluted tubules and that probenecid affected this reabsorption mechanism.

The Significance of Immunoassayable Insulin in Urine

Although a great deal of information is available regarding levels of circulating plasma insulin, not much is known of its extravascular and intracellular concentrations. A number of recent studies have measured insulin in bile, cerebrospinal fluid, and lymph, while this report is concerned with the usefulness and significance of urinary insulin in health and some disease states. There are certain advantages in measuring insulin in urine. It reflects average serum levels over an extended period of time and is useful in situations in which repeated blood sampling is impractical, such as the newborn. Measurement of urine insulin is feasible in insulin-requiring diabetic patients for their serum antibodies do not usually pass the glomerulus. Finally, urine or lymphatic insulin may be a more valid reflection of the tissue concentration than the intravascular levels. Methods Assay Technique.— Before sensitive immunologic methods were available, complicated extraction and purification techniques were necessary for the

The significance of immunoassayable insulin in urine

The significance of immunoassayable insulin in urine

Radioimmunoassay of insulin in urine.

1.Details are given of a double antibody immunoassay method suitable for measuring insulin in urine. — 2. The method proved specific and reproducible. Human insulin added to urine was quantitatively recovered and dilution and concentration resulted in proportional changes in measured insulin. — 3.Urines kept at 4°C for 24 hours showed no loss of assayable insulin, while decreases of 4–23 % were observed after standing at room temperature for 24 hours; samples of urine adjusted to pH 7.4–8.4 and centrifuged were kept at −20°C for 9 months without loss. Addition of albumin to urine to prevent adsorption of insulin on the container was found to be unnecessary. — 4. Tests of the first antibody reaction showed that non-specific co-precipitation of radioactive insulin was less than 3.5 % and that addition of sodium chloride did not alter the standard insulin curve. Monitor tubes containing131I-guinea-pig gamma-globulin showed optimal second antibody precipitation in all the urine assays surveyed. — 5. In normal subjects, the renal clearance of insulin was relatively constant over a wide range of serum insulin levels, but varied with body size. However, preliminary studies suggested that the clearance was reduced in pregnancy, hypertension and peripheral vascular disease, all of which are characterized by high serum insulin values. In contrast, the clearance in acromegaly was normal as both serum and urine insulin levels were raised. In chronic renal disease the urinary excretion of insulin was very high. — 6. Direct measurement of urinary insulin was feasible in insulin-treated diabetics provided that renal function was normal; insulin-binding antibodies were found in the urine of these diabetics only in association with proteinuria.

Evidence of the presence of insulin in urine

A fraction of urine isolated by means of ion-exchange chromatography caused enhanced uptake of glucose by rat diaphragm and epididymal fat pad, and intensified respiration of the latter. Concentrated solutions of the fraction induced hypoglycemic convulsions in mice. The yield of activity in the urine rose following administration of glucose to healthy individuals. The activity was not abolished by treatment with acids, but was abolished by alkali treatment, and by heating at 100°, as well as by thiols. On these criteria, it is concluded that the urinary fraction examined contained insulin.

The detection of insulin in urine

The detection of insulin in urine