Renal evaluation in women with preeclampsia.

BACKGROUND/AIMS: Preeclampsia (PE) is a cause of glomerulopathy worldwide. Urinary retinol-binding protein (RBP) is a marker of proximal tubular dysfunction, albuminuria is an endothelial injury marker, urine protein:creatinine ratio (PCR) may have a predictive value for renal disease later in life, and, recently, podocyturia has been proposed as a sensitive tool in pregnancy, but it needs to be tested. The aim of this study was to evaluate renal involvement in PE and healthy pregnancy. METHODS: Case-control study with 39 pregnant women assessed after 20 weeks of gestation (25 in the control group, CG, and 14 in the PE group) by performing urinary tests. RESULTS: Mean (±SD) age and gestational age of the CG were 26.9 ± 6.4 years and 37.1 ± 5.0 weeks, and of the PE group 26.4 ± 6.9 years and 30.6 ± 5.6 weeks, respectively (p = 0.001). Mean (±SD) urinary RBP (p = 0.017), albuminuria (p = 0.002), and urinary albumin concentration (UAC) ratio (p = 0.006) of the CG were 0.4 ± 0.7 mg/l, 7.3 ± 6.9 mg/l, and 8.2 ± 6.7 mg/g and of the PE group 2.0 ± 4.4 mg/l, 2,267.4 ± 2,130.8 mg/l (p = 0.002), and 3,778.9 ± 4,296.6 mg/g (p = 0.006), respectively. Mean (±SD) urine PCR in the PE group was 6.7 ± 6.1 g/g (p < 0.001). No statistical differences were found between podocyturia in the CG and PE group (p = 0.258). CONCLUSIONS: Urinary RBP, PCR, albuminuria, and UAC ratio were elevated in the PE group in comparison to the CG. Podocyturia did not predict PE.

PP019 The role of renal markers in women with and without preeclampsia: Evaluation of urinary excretion of podocytes and proteins.

INTRODUCTION: Preeclampsia (PE) is an important cause of glomerulopathy. Assessment of renal markers during pregnancy may have a predictive value for glomerular disease later in life. The early detection of PE may prevent the complications of this syndrome. OBJECTIVES: Assess the glomerular involvement in PE and in normal pregnancy by evaluating renal markers such as podocyturia and proteinuria. METHODS: Case-control study with 39 pregnant women after 20 weeks of gestation (control group - CG with n=25 and PE with n=14), we assessed podocyturia (cytospin method) and proteinuria (albuminuria, urine protein:creatinine - PCR, urinary retinol protein - RBP and albumin/creatinine ratio - ACR). (Grant FAPESP 08/56338-1) RESULTS: Mean±standard deviation of age and mean gestational age of CG were 26.9±6.4years and 37.1±5.0weeks and of PE, 26.4±6.9 and 30.6±5.6, respectively (p=0.001). No statistical differences were found between podocyturia in CG and PE although it was more frequent in this last group (p=0.258). Podocyte cells and parietal epithelial cells were detected in the slides. Mean±standard deviation of urinary RBP (p=0.017), albuminuria (p=0.002) and UAC ratio (p=0.006) of CG were 0.4±0.7mg/L, 7.3±6.9mg/L and 8.2±6.7mg/g and of PE, 2.0±4.4mg/L, 2267.4±2130.8mg/L (p=0.002) and 3778.9±4296.6mg/g (p=0.006), respectively. Mean value±standard deviation of urine PCR in PE was 6.7±6.1g/g (p=< 0.001). CONCLUSION: Urinary RBP, PCR, albuminuria and UAC ratio were elevated in PE in comparison to CG indicating its glomerular involvement but there was no correlation between those renal parameters and podocyturia. RPC and UAC ratios were good predictors of PE, but not podocyturia. Either podocyte cells as parietal epithelial cells were detected in the urine, these findings may indicate a non-invasive marker for renal disease activity but more studies are required to determine its role in PE.

PP090. The contribution of podocyturia to the evaluation of ongoing glomerular disorder in pregnant women with kidney graft (preliminary results).

INTRODUCTION: Progressive proteinuria and glomerulosclerosis characterize chronic allograft nephropathy. However, the causes are not fully elucidated. Injury of parietal epithelial cells in glomeruli, the podocytes, is the initiating cause of many renal diseases, leading to proteinuria with possible progression to glomerulosclerosis. Podocytes are highly specialized cells, with an important role in maintaining the glomerular filtration barrier and producing growth factor for mesangial cells and endothelial cells. With their foot processes they cover the glomerular basement membrane, and form slit diaphragms with neighboring podocytes. The potential role of podocytes in the failing transplanted kidney is unknown. OBJECTIVES: To evaluate podocyturia as a functional marker in pregnant women with kidney grafts. METHODS: Twenty pregnant women with kidney grafts had their urine samples cytocentrifugated and evaluated by indirect immunofluorescence. The slide was incubated for 45' at room temperature with fluorescein (FITC) anti-rabbit IgG secondary antibody (Sigma-Aldrich, EUA). Then Vectashield (mounting medium for fluorescence) with DAPI (4'6-diamino-2-fenilindol dihidrocloreto) were applied H-1200 (Vector laboratories, inc, USA). The podocytes and the total number of cells were counted in 15 fields photographed under 400x magnification with a digital camera coupled to an epifluorescence microscope DM1000 (Leica, Germany) connected to a computer. The results were expressed as podocyte/total cells (%) per area of higher cell concentration (hot spots) in the field of 400x detected by staining of nuclei and cytoplasm. (Grant FAPESP 08/56338-1). RESULTS: The mean age of the women was 26years. The urinalysis was performed at the third trimester of gestation; 11 did not exhibit urinary podocytes and 9 had podocyturia. There was also a relationship between blood pressure levels, proteinuria and the excretion of podocytes. CONCLUSION: Urinary podocyte number, blood pressure and proteinuria were associated. We observed that urinary podocyte excretion occurs in pregnant women with kidney transplant almost synchronously with higher systolic and diastolic blood pressure and higher mean levels of proteinuria. The detection of podocyturia in these women could be useful for early diagnosis and follow-up of glomerular injury, eventually preeclampsia. It may be also associated to its severity or activity, although additional studies are necessary to confirm these aspects.

Successful treatment for a case of near-fatal secondary adrenal insufficiency.

A thirty-six-year old female with shock was found to be unconsciousness a few days after developing a respiratory infection. Her past medical history included autoimmune hypothyroidism. Her state of shock was not controlled by massive fluid resuscitation with a vasopressor and antibiotics. However an infusion of 250 mg methylprednisolone dramatically improved her shock state. Further examination indicated secondary acute adrenal insufficiency. Adrenal insufficiency may complicate other endocrine disorders. Accordingly, a physician should consider hypoadrenocorticism, when patients are in a state of refractory shock in spite of massive infusion with a vasopressor especially in patients with other endocrine disorders.

Successful treatment for a case of near-fatal secondary adrenal insufficiency / Tratamiento exitoso de una caso de insuficiencia adrenal secundaria casi fatal

A thirty-six-year old female with shock was found to be unconsciousness a few days after developing a respiratory infection. Her past medical history included autoimmune hypothyroidism. Her state of shock was not controlled by massive fluid resuscitation with a vasopressor and antibiotics. However, an infusion of 250 mg methylprednisolone dramatically improved her shock state. Further examination indicated secondary acute adrenal insufficiency. Adrenal insufficiency may complicate other endocrine disorders. Accordingly, a physician should consider hypoadrenocorticism, when patients are in a state ofrefractory shock in spite ofmassive infusion with a vasopressor, especially in patients with other endocrine disorders.

Una mujer de treinta y seis años en shock fue hallada inconsciente unos dias después de desarrollar una infección respiratoria. Los antecedentes en su historia clinica incluian hipotiroidismo autoinmune. Su estado de shock no fue controlado por la reanimación con liquidos masiva con un vasopresor y antibióticos. Sin embargo, una infusion de 250 mg metilprednisolona habia mejorado considerablemente su estado de shock. Un examen mas detenido indicó insuficiencia adrenal aguda secundaria. La insuficiencia adrenal puede complicar otros trastornos endocrinos. En consecuencia, un médico debe considerar la posibilidad de hipoadrenocorticismo, cuando los pacientes se encuentran en estado de shock refractario a pesar de una infusion masiva con un vasopresor, especialmente en el caso pacientes con otros trastornos endocrinos.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels.

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54% according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 +/- 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 +/- 25.9 and 61.18 +/- 25.04 mL min-1 (1.73 m(2))-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2%) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7%) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6%) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54 percent according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 ± 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 ± 25.9 and 61.18 ± 25.04 mL min-1 (1.73 m²)-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2 percent) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7 percent) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6 percent) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

Serum leptin concentration during puberty in healthy nonobese adolescents

Data obtained during the past five years have indicated that there are important age- and gender-based differences in the regulation and action of leptin in humans. To study the physiological changes of leptin during puberty in both sexes, and its relationship with body composition and sexual maturation, we measured leptin concentrations in 175 healthy adolescents (80 girls, 95 boys, 10-18 years of age), representing all pubertal stages. We excluded individuals with a body mass index (BMI) below the 5thor above the 95th percentile relative to age. Serum concentrations of leptin were determined by a monoclonal antibody-based immunofluorimetric assay, developed in our laboratory. Body composition was determined by dual-energy X-ray absorptiometry. Pubertal stage was assigned by physical examination, according to Tanner criteria for breast development in females and genital development in males. Leptin concentration in girls (N = 80) presented a positive linear correlation with age (r = 0.35, P = 0.0012), BMI (r = 0.65, P < 0.0001) and percentfat mass (r = 0.76, P < 0.0001). In boys (N = 95) there was a positive correlation with BMI (r = 0.49, P < 0.0001) and percentfat mass (r = 0.85, P < 0.0001), but a significant negative linear correlation with Tanner stage (r = -0.45, P < 0.0001) and age (r = -0.40, P < 0.0001). The regression equation revealed that percentfat mass and BMI are the best parameters to be used to estimate leptin levels in both sexes. Thus, the normal reference ranges for circulating leptin during adolescence should be constructed according to BMI or percentfat mass to assure a correct evaluation

Serum leptin concentration during puberty in healthy nonobese adolescents.

Data obtained during the past five years have indicated that there are important age- and gender-based differences in the regulation and action of leptin in humans. To study the physiological changes of leptin during puberty in both sexes, and its relationship with body composition and sexual maturation, we measured leptin concentrations in 175 healthy adolescents (80 girls, 95 boys, 10-18 years of age), representing all pubertal stages. We excluded individuals with a body mass index (BMI) below the 5th or above the 95th percentile relative to age. Serum concentrations of leptin were determined by a monoclonal antibody-based immunofluorimetric assay, developed in our laboratory. Body composition was determined by dual-energy X-ray absorptiometry. Pubertal stage was assigned by physical examination, according to Tanner criteria for breast development in females and genital development in males. Leptin concentration in girls (N = 80) presented a positive linear correlation with age (r = 0.35, P = 0.0012), BMI (r = 0.65, P < 0.0001) and %fat mass (r = 0.76, P < 0.0001). In boys (N = 95) there was a positive correlation with BMI (r = 0.49, P < 0.0001) and %fat mass (r = 0.85, P < 0.0001), but a significant negative linear correlation with Tanner stage (r = -0.45, P < 0.0001) and age (r = -0.40, P < 0.0001). The regression equation revealed that %fat mass and BMI are the best parameters to be used to estimate leptin levels in both sexes. Thus, the normal reference ranges for circulating leptin during adolescence should be constructed according to BMI or %fat mass to assure a correct evaluation.

Urinary retinol-binding protein as a prognostic marker in the treatment of nephrotic syndrome.

We studied the urinary levels of retinol-binding protein (urRBP), an index of proximal tubular dysfunction, in patients with nephrotic syndrome before and approximately 2 months after the beginning of steroid therapy as a predictor of response to therapy which included for some patients courses of immunosuppressive drugs. Those patients with minimal-change disease, mesangial proliferative glomerulonephritis, and focal-segmental glomerulosclerosis who had normal pretreatment urRBP levels were responsive to treatment; occasionally, responsive patients had an initially elevated urRBP level which normalized during treatment. Contrariwise, those patients with abnormally high levels of urRBP which did not normalize during treatment did not respond to treatment. The chance of a patient with minimal-change disease, mesangial proliferative glomerulonephritis, or focal-segmental glomerulosclerosis and a pretreatment urRBP level equal to or >1.0 mg/l being resistant to steroid treatment is 30 times that of a patient with a urRBP level <1.0 mg/l and even higher, if we consider the levels obtained during treatment.

[Retinol-binding protein urinary levels in a pediatric population: evolution according to age] / Níveis urinários da proteína transportadora do retinol em uma população pediátrica: evolução em função da idade.

OBJECTIVE: The kidney of the child, mainly the newborn, is specially susceptible to ischemic and/or toxic injuries, which can affect the renal tubular function. Therefore, reliable and precocious markers of tubular dysfunction would be useful in Pediatrics. The urinary levels of retinol binding-protein (RBPu) has been used in this way. Our goal was to evaluate the RBPu behavior in childhood to establish a reference interval and verify possible differences according to the age. METHODS: We studied healthy children aged 1 month to 8 years and healthy term and preterm babies; in the last group we only included babies without hemodynamic repercussion. We collected isolated urine samples once for children above 1 month and periodically for those below this age. In this urine, besides the dipsticks test (Combur 9(R), Boehringer), RBPu and creatinine were measured. RESULTS: We observed that the RBPu levels in childhood do not have a normal distribution; after logarithm data transformation we were able to establish an interval of reference for children above one month of age: 3 to 269 micro g/L, mean of 27 micro g/ L. In newborns, we found a large variation of RBPu excretion. However, we noticed that the excretion decreases significantly, reaching the healthy adult levels at the end of the 1st month, for term as well as for preterm newborns. CONCLUSIONS: We suggest that RBPu can be used as a marker of proximal tubular dysfunction, since we could establish an interval of reference, and it can be repeated whenever necessary in the follow-up of this kind of injury, because it is not an invasive exam. Moreover, this study contributes to the knowledge of the tubular function development for showing that at the end of the 1st month of life the newborn's kidney is able to reabsorb RBPu as the healthy adult kidney is.

A specific and highly sensitive time-resolved fluoroimmunoassay for human proinsulin.

We describe a time-resolved fluoroimmunoassay specific for human proinsulin using a combination of two high-affinity monoclonal antibodies, one against insulin and the other specific for intact proinsulin and for split 65-66 and des 64-65 proinsulin forms. The assay employs only 200 microl of serum, with a detection limit of 0.1 pmol/l. The intra-assay variation coefficient was less than 3% between 3 and 1000 pmol/l. There was 0% cross-reaction with insulin, C-peptide, split 32-33 and des 31-32 proinsulin. Serum concentration of proinsulin was analyzed in 50 subjects during an oral glucose tolerance test (10 non-obese control, 10 obese controls, 10 subjects with impaired glucose tolerance, 10 patients with type II diabetes mellitus (DM) and fasting blood glucose (FBG) < 140 mg/dl, and 10 patients with type II DM and FBG > 150 mg/dl). Mean fasting serum proinsulin levels measured by this assay in non-obese controls (0.84 - 0.90 pmol/l; 0.1-2.4 pmol/l) were lower than the results reported by other investigators. There was an increase of proinsulin related to obesity and increased glucose levels, suggesting that proinsulin levels increase with insulin resistance.

A specific and highly sensitive time-resolved fluoroimmunoassay for human proinsulin

We describe a time-resolved fluoroimmunoassay specific for human proinsulin using a combination of two high-affinity monoclonal antibodies, one against insulin and the other specific for intact proinsulin and for split 65-66 and des 64-65 proinsulin forms. The assay employs only 200 micro liters of serum, with a detection limit of 0.1 pmol/l. The intra-assay variation coefficient was less than 3 percent between 3 and 1000 pmol/l. There was 0 percent cross-reaction with insulin, C-peptide, split 32-33 and des 31-32 proinsulin. Serum concentration of proinsulin was analyzed in 50 subjects during an oral glucose tolerance test (10 non-obese controls, 10 obese controls, 10 subjects with impaired glucose tolerance, 10 patients with type II diabetes meIlitus (DM) and fasting blood glucose (FBG) <140 mg/dl, and 10 patients with type II DM and FBG >150 mg/dl). Mean fasting serum proinsulin levels measured by this assay in non-obese controls (0.84 +/-0.90 pmol/l; 0.1-2.4 pmol/l) were lower than the results reported by her investigators. There was an increase of proinsulin related to obesity and increased glucose levels, suggesting that proinsulin levels increase with insulin resistance.

Monoclonal antibodies specific for the free alpha subunit of glycoprotein hormones and their use in the development of a sensitive immunofluorometric assay.

Glycoprotein hormone free alpha subunit has been used as a marker for some pituitary tumors and to study the reactivity of glycoprotein hormone-producing cells under different circumstances. We describe a highly sensitive and specific immunofluorometric assay for the measurement of serum free alpha subunit levels. The assay is based on a monoclonal antibody, specific for free alpha subunit, bound to microtiter plates. As tracer antibody we employed an europium-labelled free/complexed alpha subunit specific monoclonal antibody. Using overnight incubation and 50-microliters samples, the least detectable dose was of the order of 4 ng/l. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively. Normal adult males showed values ranging from 120 to 790 ng/l, not different from normal adult premenopausal females (88 to 604 ng/l). In post-menopausal females, serum concentrations were significantly higher, ranging from 341 to 4071 ng/l. In 56 patients with untreated pituitary tumors (18 "non-secreting", 25 GH-producing and 13 prolactin-producing tumors), 10 showed high values, 3 of them from the first group, 3 from the second and 4 from the third. We conclude that this highly sensitive assay can be a valuable tool for the diagnosis and follow-up of selected patients with pituitary tumors and in other circumstances in which the glycoprotein hormone-producing cells of the pituitary require evaluation.

Monoclonal antibodies specific for the free alpha subunit of glycoprotein hormones and their use in the development of a sensitive immunofluorometric assay

Glicoprotein hormone free alpha subunit has been used as a marker for some pituitary tumors and to study the reactivity of glycoprotein hormone-producing cells under different circunstances. We describe a highly sensitive ans specific immunofluorometric assau for the measurement of serum free alpha subunit levels. The assay is based on a monoclonal antibody, specific for free alpha subunit, bound to microtiter plates. As tracer antibody we employed an europium-labelled free/complexed alpha subunit specific monoclonal antibody. Using overnight incubation and 50µl samples, the least detectable dose was of the order of 4 ng/1. Cross-reactivity with LH, TSH, FSH, and hCG was 6.5, 1.2, 4.3 and 1.1 percent, repectively. Normal adult males showed values ranging from 120 to 790ng/l, not different from normal adult premenopausal females (88 to 604 ng/l). In post-menopausal females, serum concentrations were significantly highler, ranging from 341 to 407 ng/l. In 56 patients with untreated pituitary tumors (18 "non-secreting", 25 GH-producing and 13 prolactin-producing tumors), 10 showed high values, 3 of them from the first group, 3 from the second and 4 from the third. We conclude that this highly sensitive assay can be a valualbe tool for the diagnosis and follow-up of selected patients with pituatary tumors and in other circumstances in which the glycoprotein hormone-producing cells of the pituitary require evaluation

Comparison of the determination of insulin by a monoclonal antibody-based immunofluorometric assay and by radioimmunoassay.

This paper describes an immunofluorometric assay (IFMA) for insulin and compares it with the classical radioimmunoassay (RIA). Monoclonal antibodies against insulin were produced and used to develop the IFMA. One, immobilized on microtiter plates, was used for capture, the other, labelled with Europium, was used as tracer antibody. The IFMA presents sensitivity to an amount of insulin of 3 pmol/l and acceptable values for intra- and interassay error. The IFMA presented superimposable curves for human insulin, Arg65/Gly66-split proinsulin and des-Lys64,Arg65, and no cross-reactivity with human proinsulin, Arg32/Glu33-split and des-Arg31,Arg32. The RIA showed 100% cross-reactivity with human proinsulin, 90% with Arg32/Glu33-split, 193% with Arg65/Gly66-split, 340% with des-Arg31,Arg32 and 170% with des-Lys64,Arg65. The assays were used to measure insulin in 300 serum samples from 50 subjects submitted to an oral glucose tolerance test (OGTT). Twenty were normal, 10 had impaired glucose tolerance and 20 non-insulin-dependent diabetes mellitus. The mean value (+/- SEM) obtained by IFMA was 166.7 +/- 12.1 pmol/l and the mean value obtained by RIA was 339.6 +/- 18.6, with a correlation of r = 0.80 (P < 0.01). Comparison of basal insulin levels of the different groups of individuals using IFMA or RIA led to the same conclusions. The area under the curve showed statistically significant differences only for the comparison between normal lean subjects and individuals with impaired glucose tolerance, when measured by RIA. Our data stress the importance of methodology definition when comparing insulin results.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the determination of insulin by a monoclonal antibody-based immunofluorometric assay and by radioimmunoassay

This paper describes an immunofluorometric assay (IFMA) for insulin and compares it with the classical radioimmunoassay (RIA). Monoclonal antibodies against insulin were produced and used to develop the IFMA. One, immobilized on microtiter plates, was used for capture, the other, labelled with Europium, was used as tracer antibody. The IFMA presentes sensitivity to an amount of insulin of 3 pmol/1 and acceptable valueus for intra- and interassay error. The IFMA presented superimposable curves for human insulin, Arg65/Gly66-split proinsulin and des-Lys64, Arg65, and no cross-reactivity with human proinsulin, Arg32/Glu33 -split and des-Arg31, Arg32. The RIA showed 100 percent cross-reactivity with human proinsulin, 90 pecent with des-Arg31, Arg32 and 170 percent with des-Lys64, Arg65. The assay were used to measure insulin in 300 serum samples from 50 subjects submitted to an oral glucose tolerance test (OGTT). Twenty were normal, 10 had impaired glucose tolerance and 20 non-insulin-dependent diabetes mellitus. The mean value (ñ SEM) obtained bu IFMA was 166.7 ñ 12.1 pmol/1 and the mean value obtained by RIA was 339.6 ñ 18.6, with a correlacion of r = 0.80 (P0.01). Comparison of basal insulin levels of the different groups of individuals using IFMA or RIA led to the same conclusions. The area under curve showed statistically significant differences only for the comparison between normal lean subjects and individuals with impaired glucose tolerance, when measured by RIA...(au)