Detection of recombinant insulins in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry after immunoaffinity purification based on monolithic microcolumns.

This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography-electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra®, Humalog®, Levemir®, NovoRapid®, and Tresiba®) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02-0.05 ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba® and/or Humalog®, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. Graphical abstract A hybrid assay comprising MSIA-based immunoextraction combined with liquid chromatography-electrospray tandem mass spectrometry was developed and validated for the detection of recombinant insulins in human urine.

Polyphenol-rich curry made with mixed spices and vegetables benefits glucose homeostasis in Chinese males (Polyspice Study): a dose-response randomized controlled crossover trial.

PURPOSE: To investigate acute effects of two doses of a polyphenol-rich curry made with seven different spices and four base vegetables, eaten with white rice, on 24 h glucose response, postprandial insulinemia, triglyceridemia and 24 h urinary total polyphenol excretion (TPE). METHODS: Randomized, controlled, dose-response crossover trial in healthy, Chinese men [n = 20, mean ± standard deviation (SD) age 23.7 ± 2.30 years, BMI 23.0 ± 2.31 kg/m2] who consumed test meals matched for calories, macronutrients and total vegetables content, consisting either Dose 0 Control (D0C) or Dose 1 Curry (D1C) or Dose 2 Curry (D2C) meal. 24 h glucose concentration was measured using continuous glucose monitoring (CGM), together with postprandial plasma insulin and triglyceride for up to 7 h. Total polyphenol content (TPC) of test meals and urinary TPE were measured using the Folin-Ciocalteu assay. RESULTS: TPC for D0C, D1C and D2C were 130 ± 18, 556 ± 19.7 and 1113 ± 211.6 mg gallic acid equivalent (GAE) per portion served, respectively (p < 0.0001). Compared with D0C meal, we found significant linear dose-response reductions in the 3-h postprandial incremental AUC (iAUC) for CGM glucose of 19% and 32% during D1C and D2C meals respectively (p < 0.05) and non-significant linear dose response reductions in iAUC of insulin (p = 0.089). Notably, we found significant dose-dependent increases in postprandial triglyceride with increasing curry doses (p < 0.01). Significant increases in TPE with increasing curry doses were also observed (p < 0.01). CONCLUSION: Polyphenol-rich curry intake can improve postprandial glucose homeostasis. The longer term effects remain to be established.

Current trends in mass spectrometry of peptides and proteins: Application to veterinary and sports-doping control.

Detection of misuse of peptides and proteins as growth promoters is a major issue for sport and food regulatory agencies. The limitations of current analytical detection strategies for this class of compounds, in combination with their efficacy in growth-promoting effects, make peptide and protein drugs highly susceptible to abuse by either athletes or farmers who seek for products to illicitly enhance muscle growth. Mass spectrometry (MS) for qualitative analysis of peptides and proteins is well-established, particularly due to tremendous efforts in the proteomics community. Similarly, due to advancements in targeted proteomic strategies and the rapid growth of protein-based biopharmaceuticals, MS for quantitative analysis of peptides and proteins is becoming more widely accepted. These continuous advances in MS instrumentation and MS-based methodologies offer enormous opportunities for detection and confirmation of peptides and proteins. Therefore, MS seems to be the method of choice to improve the qualitative and quantitative analysis of peptide and proteins with growth-promoting properties. This review aims to address the opportunities of MS for peptide and protein analysis in veterinary control and sports-doping control with a particular focus on detection of illicit growth promotion. An overview of potential peptide and protein targets, including their amino acid sequence characteristics and current MS-based detection strategies is, therefore, provided. Furthermore, improvements of current and new detection strategies with state-of-the-art MS instrumentation are discussed for qualitative and quantitative approaches.

Elevated glucose levels in early puerperium, and association with high cortisol levels during parturition.

Background Gestational diabetes is one of the commonest metabolic problems associated with pregnancy and an accurate diagnosis is critical for the care. Research has shown that pregnant women have high levels of cortisol during the last stage of parturition. As cortisol is a diabetogenic hormone causing increased glucose levels, we wanted to study the association between cortisol and glucose levels during parturition. Materials and methods Glucose and cortisol were analyzed during parturition in 50 females divided according to slow (n = 11) and normal labors (n = 39). Blood samples were analyzed three times during the parturition and four times in the first day after delivery. Glucose levels were also measured once in each trimester. Results In the normal group, the glucose concentration increased from 6.2 (IQR 5.6-8.0) mmol/L in the latency phase to 11.6 (10.0-13.3) mmol/L at aftercare (p < 0.05). After parturition the glucose concentrations decreased gradually. There were significant Spearman rank correlations between glucose and cortisol values. Conclusions The changes associated with birth cause significant elevations of cortisol and glucose around parturition.

Sensitive detection of human insulin using a designed combined pore approach.

A unique combined pore approach to the sensitive detection of human insulin is developed. Through a systematic study to understand the impact of pore size and surface chemistry of nanoporous materials on their enrichment and purification performance, the advantages of selected porous materials are integrated to enhance detection sensitivity in a unified two-step process. In the first purification step, a rationally designed large pore material (ca. 100 nm in diameter) is chosen to repel the interferences from nontarget molecules. In the second enrichment step, a hydrophobically modified mesoporous material with a pore size of 5 nm is selected to enrich insulin molecules. A low detection limit of 0.05 ng mL(-1) in artificial urine is achieved by this advanced approach, similar to most antibody-based analysis protocols. This designer approach is efficient and low cost, and thus has great potential in the sensitive detection of biomolecules in complex biological systems.

Association of infertile patients having polycystic ovarian syndrome with recurrent miscarriage.

Polycystic ovarian syndrome (PCOS) has a pivotal role in the development of various complications during pregnancy. Polycystic ovarian syndrome women having elevated LH and hyper insulineuia may be at increased risk of miscarriage. The study was done to find out the recurrent pregnancy loss among the PCOS patient. This was a cross sectional case control study in total 100 infertile patients between age 20-40 years attending BSMMU out patient Department from July 2011 to June 2012, among them 50 infertile patients with PCOS regarding as a case and 50 infertile patients without PCOS selected as a control. Regarding case (infertile patients with PCOS) shows 20(40%) recurrent miscarriage and among control (infertile patients without PCOS) shows recurrent miscarriage 6(12%). And also among case group shows insulin resistance 8(16%) and control group insulin resistance 1(2%). Six (75%) abortion occur among PCOS with insulin resistance and 5(62.5%) abortion occur among PCOS with raised testosterone level. It is observed that recurrent miscarriage is higher in PCOS group. And also concluded that insulin resistance and raised testosterone level is responsible for this condition. So, further large scale study would be needed to reduce the chance of recurrent pregnancy loss by treatment with insulin sensitizer in case of obese PCOS with insulin resistance patient.

Insulin.

Due to its versatile nature and its corresponding anabolic and anticatabolic properties, insulin has been prohibited in sports since 1999. Numerous studies concerning its impact on glycogen formation, protein biosynthesis, and inhibition of protein breakdown have illustrated its importance for healthy humans and diabetics as well as elite athletes. Various reports described the misuse of insulin to improve performance and muscle strength, and synthetic analogs were the subject of several studies describing the beneficial effects of biotechnologically modified insulins. Rapid- or long-acting insulins were developed to enhance the injection-to-onset profile as well as the controllability of administered insulin, where the slightest alterations in primary amino acid sequences allowed the inhibition of noncovalent aggregation of insulin monomers (rapid-acting analogs) or promoted microprecipitation of insulin variants upon subcutaneous application (long-acting analogs). Information on the metabolic fate and renal elimination of insulins has been rather limited, and detection assays for doping control purposes were primarily established using the intact compounds as target analytes in plasma and urine specimens. However, recent studies revealed the presence of urinary metabolites that have been implemented in confirmation methods of sports drug testing procedures. So far, no screening tool is available providing fast and reliable information on possible insulin misuse; only sophisticated procedures including immunoaffinity purification followed by liquid chromatography and tandem mass spectrometry have enabled the unambiguous detection of synthetic insulins in doping control blood or urine samples.

Urinary detection of rapid-acting insulin analogs in healthy humans.

Human insulin and its synthetic analogs are considered as life-saving drugs for people suffering from diabetes mellitus. Next to the therapeutic use, scientific and non-scientific literature (e.g. bodybuilding forums; antidoping intelligence and investigation reports) indicate that these prohibited substances are used as performance enhancing agents. In the present report, the development and validation of a sensitive analytical strategy is described for the urinary detection of three rapid-acting insulin analogs (Lispro, Aspart, Glulisine). The method is based on sample purification by the combination of ultrafiltration and immunoaffinity purification and subsequent analysis by nano-flow liquid chromatography coupled to high resolution mass spectrometry. Next to the results on different validation parameters (LOD: 10 pg/mL; recovery: 25-48%; matrix effect: -3-(-8) %), data on urinary elimination times, which were obtained in the frame of an administration study with the participation of healthy volunteers, are presented. The determined detection windows (~9 hours) are expected to help to evaluate current routine analytical methods and aim to aid doping authorities to set appropriate target windows for efficient testing.

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.

The uridine diphosphate glucuronosyltransferase 2B15 D85Y and 2B17 deletion polymorphisms predict the glucuronidation pattern of androgens and fat mass in men.

CONTEXT: Previous in vitro studies have demonstrated that the UDP glucuronosyltransferase (UGT)2B15 and UGT2B17 glucuronidate androgens and their metabolites. OBJECTIVE: Our objective was to determine in vivo whether the UGT2B15 D85Y and the UGT2B17 deletion polymorphisms predict androgen glucuronidation and body composition. PARTICIPANTS: Two population-based cohorts including young adult (n = 1068; age = 18.9 yr) and elderly (n = 1001; age = 75.3 yr) men were included in the study. MAIN OUTCOME MEASURES: Serum and urine levels of testosterone (T) and dihydrotestosterone (DHT) were measured by gas chromatography-mass spectrometry, and serum levels of the major glucuronidated androgen metabolites androstane-3alpha,17beta-diol(androstanediol)-3-glucuronide, androstanediol-17-glucuronide, and androsterone-glucuronide were measured by liquid chromatography-tandem mass spectrometry. Body composition was measured by dual-energy x-ray absorptiometry. RESULTS: Both the UGT2B15 D85Y and the UGT2B17 deletion polymorphisms were associated with serum levels of androstanediol-17-glucuronide (P < 0.001) but not with levels of androstanediol-3-glucuronide or androsterone-glucuronide in both cohorts. Glucuronidation of T and DHT was associated with the UGT2B17 deletion but not with the UGT2B15 D85Y polymorphism, suggested by strong associations between the deletion polymorphism and urine levels of these two hormones. Both polymorphisms were associated with several different measures of fat mass (P < 0.01). The UGT2B17 deletion polymorphism was associated with insulin sensitivity (P < 0.05) as indicated by the homeostasis model assessment index. CONCLUSIONS: The UGT2B15 D85Y and the UGT2B17 deletion polymorphisms are both predictors of the glucuronidation pattern of androgens/androgen metabolites. Our findings indicate that UGT2B17 is involved in 17-glucuronidation of mainly T but also of DHT and androstanediol and that UGT2B15 is involved in the 17-glucuronidation of androstanediol. Furthermore, these two polymorphisms are predictors of fat mass in men.

The renal handling of insulin.

The renal handling of insulin was studied by insulin immunoassay in arterial blood, renal venous blood, and urine of fasting patients with normal renal function and in peripheral venous blood and urine of normal subjects and patients with renal disease before and after an oral glucose load. A renal arteriovenous insulin concentration difference of approximately 29% was found and suggests that in normal subjects renal insulin clearance is significantly in excess of glomerular filtration rate. The insulin excreted in the urine of normal individuals at no time exceeded 1.5% of the load filtered at the glomerulus. This contrasts with the finding of a urinary insulin clearance approaching glomerular filtration rate in patients with severely impaired renal tubular function. It is suggested that insulin is normally filtered at the glomerulus and then almost completely reabsorbed or destroyed in the proximal tubule. If reabsorption occurs, as seems more likely, reabsorbed insulin does not return to the renal vein and is presumably utilized in renal metabolism together with insulin taken up directly from the blood. Caution is advised in the use of urinary insulin concentration or excretion as an index of serum insulin level or insulin secretion because a very small and variable proportion of filtered insulin appears in the urine in normal subjects, and major changes in urinary insulin excretion may arise as a result of minor tubular defects.

Metabolism of proinsulin, insulin, and C-peptide in the rat.

The renal extraction and excretion of bovine proinsulin, insulin, and C-peptide and the contribution of the kidney to their total metabolic clearance rate (MCR) were studied in the rat. Metabolic clearance rates were measured by the constant infusion technique and plasma and urine concentrations of each polypeptide were determined by radioimmunoassay. The MCR of insulin (16.4+/-0.4 ml/min) was significantly greater than that of either proinsulin (6.7+/-0.3 ml/min) or C-peptide (4.6+/-0.2 ml/min). Metabolic clearance rates were independent of plasma levels over a range of steady-state plasma concentrations varying from 1 to 15 ng/ml.In contrast to the differences in their metabolic clearance rates, the renal disposition of the three polypeptides was similar, being characterized by high extraction and very low urinary clearance. The renal arteriovenous difference of proinsulin, insulin, and C-peptide averaged 36, 40, and 44%, respectively, and was linearly related to their arterial concentration between 2 and 25 ng/ml. When glomerular filtration was markedly reduced or stopped by ureteral obstruction, the renal extraction of proinsulin, insulin, and C-peptide was invariably greater than the simultaneously measured extraction of inulin, indicating that these polypeptides are removed from the renal circulation by both glomerular filtration and direct uptake from peritubular capillary blood. The fractional urinary clearance of each polypeptide never exceeded 0.6%, indicating that more than 99% of the amount filtered was sequestered in the kidney. The renal removal of proinsulin and C-peptide from the circulation accounts for 55 and 69% of their metabolic clerance rates, while the renal contribution to the peripheral metabolism of insulin was smaller, averaging 33%. This difference is due to the fact that insulin, but not the other two polypeptides, is metabolized to a significant extent by the liver. These results define the renal handling of proinsulin, insulin, and C-peptide in the rat and indicate that in this species the kidney represents a major site for insulin metabolism and is the main organ responsible for the degradation of proinsulin and C-peptide.

Pyrazinoate excretion in the chimpanzee. Relation to urate disposition and the actions of uricosuric drugs.

These experiments were designed to define the renal disposition of pyrazinoic acid in a nonhuman primate that is phylogenetically close to man and to relate this to the effects of pyrazinoate on urate excretion. The renal clearance of pyrazinoate was almost always greater than the simultaneous glomerular filtration rate at plasma concentrations ranging from 1.9 to 960 mug/ml. Some inhibitors of tubular secretion, probenecid, MK-282 (an experimental, potent uricosuric drug), p-aminohippurate, iodopyracet, sulfinpyrazone, and mersalyl, reduced clearances of pyrazinoate to values far below filtration rate. Chlorothiazide, allopurinol, and salicylate did not. The clearance of pyrazinoate was not influenced by changes in urine flow. It is concluded that pyrazinoate is actively secreted and actively reabsorbed. Pyrazinoate had a dual effect on urate excretion. At concentrations in plasma less than 10 mug/ml there was a concentration related fall in urate/inulin clearance ratio, reaching values of 10-20% of control. Over the range of 10-100 mug/ml in plasma, the clearance of urate remained maximally depressed. At higher concentrations of pyrazinoate there was a concentration related increase in urate/inulin clearance ratio such that at pyrazinoate levels above 600 mug/ml a definite uricosuric response was obtained. Prior administration of pyrazinoate to give plasma levels of 20-140 mug/ml completely or almost completely prevented uricosuric responses to probenecid, PAH, chlorothiazide, and sulfinpyrazone. Iodopyracet, mersalyl, salicylate and N-acetyl-4-dibutylsulfamoyl-3-trifluoromethylbenzenesulfonamide (MK-282) retained significant uricosuric action, but the activities were probably less than normal. The results are consistent with a model of urate transport involving high rates of bidirectional transtubular flux.

Impaired metabolic profile is a predictor of capillary rarefaction in a population of hypertensive and normotensive individuals.

Capillary rarefaction is typically encountered in essential hypertension, yet identification of factors interfering with this phenomenon remains substantially underinvestigated. We examined whether components of metabolic profile (dyslipidemia, insulin resistance), inflammatory (high-sensitivity C-reactive protein, high-sensitivity C-reactive protein), and angiogenic (vascular endothelial growth factor) factors are implicated in this phenomenon in a population of newly diagnosed, never-treated hypertensive patients and normotensive controls. Nailfold capillary density was estimated with nailfold capillaroscopy using specifically designed software. A total of 159 individuals, 93 hypertensives, and 66 normotensives were included. Nailfold capillary density was lower among hypertensives compared to normotensives (146.4 ± 31.0 vs. 155.4 ± 26.9, respectively; P = .047). In the total population, capillary density significantly correlated with high-density lipoprotein (HDL) (r = 0.232; P = .003), HDL/low-density lipoprotein ratio (r = 0.175; P = .025), age (r = 0.236; P = .003), but neither with vascular endothelial growth factor or high-sensitivity C-reactive protein. An inverse association was found with body mass index (r = -0.174; P = .029), insulin levels (r = -0.200; P = .018), and homeostasis model assessment-insulin resistance (r = -0.223; P = .009). In the separate analysis for the hypertensive population, sex (P = .014) and homeostasis model assessment-insulin resistance (P = .011) were identified as significant predictors of capillary rarefaction after adjustment for other factors. On the contrary, only HDL levels (P = .036) predicted capillary density in the multiple regression model for the normotensive population. Different aspects of impaired metabolic profile, that is, insulin resistance and low HDL levels, but not angiogenic or inflammatory markers, appear to be independently associated with capillary rarefaction in hypertensive and normotensive individuals.

Reevaluation of urine C-peptide as measure of insulin secretion.

Urine C-peptide (UCP) has been proposed as a measure of insulin secretion, because insulin and C-peptide are consecreted in equimolar concentrations by the pancreatic beta-cell. The validity of this approach was tested by comparing insulin secretion rates, calculated by application of a two-compartmental analysis of peripheral C-peptide concentrations, with UCP excretion rates. Insulin secretion and UCP excretion with subjects on a mixed diet were simultaneously measured over a 24-h period in 13 patients with noninsulin-dependent diabetes mellitus and in 14 matched nondiabetic control subjects. The fraction of secreted C-peptide that was excreted in the urine (fractional C-peptide excretion) showed considerable intersubject variability in the diabetic (11.3 +/- 1.6%, range 3.9-20.8) and control (8.0 +/- 1.7%, range 1.1-27.9, P = .07) subjects (means +/- SE). UCP clearance demonstrated a similar degree of variability and was not significantly different (P = .07) between diabetic (23.8 +/- 3.0 ml/min) and control (16.5 +/- 2.7 ml/min) subjects. In control subjects, the 24-h insulin secretion rate correlated more closely with the fasting insulin secretion rate (r = .97, P = .0001), fasting C-peptide (r = .81, P = .0005), and fasting insulin (r = .80, P = .0005) concentrations than with the 24-h UCP excretion rate (r = .62, P = .02). Similar results were obtained in the diabetic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of anti-insulin receptor antibodies on in vivo insulin metabolism.

The effects of anti-insulin receptor antibodies (AIRA) on receptor binding and insulin metabolism were studied in two patients with the type B, severe insulin resistance syndrome. Insulin binding was determined using rat hepatocytes in primary culture and the patient's own red blood cells. Plasma and urinary insulin concentrations and metabolic clearance rates (MCR) were determined in the two patients and in four normal controls in response to infusions of insulin for 60-120 min at rates ranging from 1 to 925 mU/kg/min. In patient 1, basal insulin concentration was 1400 microU/ml. After infusion of 1, 10, and 925 mU/kg/min of insulin it rose to 3800, 5500, and 225,000 microU/ml, respectively. Respective MCRs were 19, 110, and 186 ml/min. In patient 2, basal insulin concentration was 440 microU/ml. After infusion of 1, 10, and 100 mU/kg/min of insulin it rose to 720, 2500, and 18,800 microU/ml, respectively. Respective MCRs were 193, 262, and 294 ml/min. In controls, basal insulin concentration was 4 +/- 0.3 microU/ml. After infusion of 1 and 10 mU/kg/min of insulin, it rose to 82 +/- 17 and 1288 +/- 50 microU/ml. Respective MCRs were 950 and 630 ml/min. These data showed that, in patients with AIRA: (1) insulin metabolism took place at the same rate but at higher insulin concentrations than in normal controls, and (2) MCR increased with rising insulin concentration but remained subnormal even at the highest insulin concentrations. In contrast, MCR in normal controls decreased with increasing insulin concentrations. The data suggest that prevention of insulin binding prevents insulin metabolism at physiologic insulin concentrations and that supraphysiologically elevated insulin concentrations are needed to activate nonreceptor mechanisms.

Renal wastage of insulin in children with diabetes mellitus.

In normal human beings the percentage of serum insulin excreted in the urine is constant over a wide range of values. The quantity of immunoreactive insulin found in the urine is believed to reflect the level of free insulin in the serum. Immunoreactive insulin was measured in the urine of nondiabetic children and diabetic children receiving exogenous insulin. Children with diabetes mellitus excreted greater amounts of immunoreactive insulin (18.5+/-8 muU./mg. creatinine) than did nondiabetic children (11.9+/-5 muU./mg. creatinine). This difference was statistically significant (p less than 0.0005). Children with "poor glycemic control" excreted a greater portion of their administered insulin dose than did those with "good control." The renal wastage of insulin correlated (r=0.94) with the duration of insulin treatment but not with the quantity administered. Antibody binding of serum insulin may explain in part these observations, but an acquired defect in the renal tubular reabsorption of insulin may also exist. Modifications in the management of diabetes that reduce the renal wastage of insulin may improve the metabolic stability of children with "poor diabetic control."

Reduced urinary insulin clearance in patient with abnormal insulinemia.

We recently reported a new case of abnormal insulinemia with LeuA3 insulin. Herein, we measured urinary insulin clearance during oral glucose tolerance tests in proband with abnormal insulinemia (44-yr-old female), three affected family members, two unaffected family members, two other hyperinsulinemic patients with obesity, five non-insulin-dependent diabetic patients, and five normal control subjects. Urinary insulin-to-creatinine clearance ratio in the proband and her affected family members was 0.22 X 10(-3) +/- 0.07 (mean +/- SD, n = 4) and was markedly reduced compared with those of other groups: 1.73 X 10(-3) in two unaffected family members, 2.77 X 10(-3) in two other hyperinsulinemic patients with obesity, 2.99 X 10(-3) +/- 1.48 in five non-insulin-dependent diabetic patients, and 2.54 X 10(-3) +/- 0.67 in five normal control subjects. In contrast, urinary C-peptide clearance in these groups was not significantly different from controls. Binding of immunopurified insulins extracted from urine of the patients with abnormal insulinemia to guinea pig kidney membrane was slightly decreased (71% of standard insulin), in contrast with the observation that serum insulin of the proband had much less receptor-binding activity. Reverse-phase HPLC analysis of the immunopurified insulin of the proband revealed that the ratios of normal insulin to abnormal insulin were 8:3 in urine and 1:7 in serum, respectively. These results suggest that excretion of abnormal insulin in urine is much less than that of normal insulin.

Insulin-like insulinase-resistant material, distinguishable from normal insulin, in juvenile diabetes.

This study compares some properties of the immunoreactive insulin-like material extracted from the urine of children with overt diabetes with that from normal children. Insulin-like species were fractionated by gel filtration and by isoelectric focusing and were tested for sensitivity to an insulin-specific degradative enzyme. Insulin concentration was measured by radioimmunoassay. The major insulin-like component from the urine of ten normal children and fifteen untreated juvenile diabetics and from the urine of four and the serum of one latent diabetics behaved (on gel filtration) as normal insulin, was sensitive to insulinase, and (in all cases studied) had an identical isoelectric point (resolution 0.1 pH units). A proportion of the immunoreactivity extracted from urine (0-4 per cent from normal children, 5-30 per cent from twelve of the thirteen nonobese untreated diabetic children) eluted from the gel filtration column before insulin. This material from diabetic urine was of two size classes, "proinsulin-like" and "mid-insulin," both resistant to degradation by insulinase. Insulinase-resistant immunoreactivity from one patient was analyzed by isoelectric focusing. Urine samples from two obese children with overt diabetes and four children with latent diabetes contained normal proportions (less than 4 per cent) of immunoreactive species larger than insulin. The possible nature and significance of the present insulinase-resistant species are briefly considered.

Early pregnancy maternal urinary metabolomic profile and later insulin resistance and fetal adiposity.

OBJECTIVE: Our objective was to determine if early pregnancy urinary metabolomic profiles could predict fetal adiposity and macrosomia. METHODS: This is a prospective study of 50 healthy women in their second pregnancy. Fasting urine samples taken during early pregnancy were analyzed using NMR spectroscopy. Maternal glucose and insulin were measured in early pregnancy and at 28 weeks and the HOMA index for insulin resistance calculated. At 34 weeks ultrasound assessed fetal anthropometry including fetal anterior abdominal wall width (AAW). At delivery birth weight was recorded. Probabilistic principal component with covariates analysis (PPCCA), a novel extension of principal component analysis, which facilitates joint modeling of metabolomic data and additional covariate information, was employed to analyze the data. RESULTS: This analysis revealed that maternal HOMA and AAW significantly covaried with the (1)H NMR derived metabolomic profile of the urine. As such, in this cohort of healthy, non-diabetic women, early pregnancy urinary metabolomic profile differed significantly according to both maternal insulin resistance and fetal fat deposition in utero. CONCLUSION: These findings hold potential for early pregnancy identification of those at risk of fetal macrosomia.