Unextracted Plasma Research Articles

Immunoreactive (IR) cholecystokinin (CCK) in unextracted human plasma represents larger molecular species which can be converted by enzyme action

Immunoreactive (IR) cholecystokinin (CCK) in unextracted human plasma represents larger molecular species which can be converted by enzyme action

Serum 17α-hydroxyprogesterone in infants and children as measured by a direct radioimmunoassay kit

A direct 17 alpha-hydroxyprogesterone (17-OHP) radioimmunoassay kit was used for the assay of samples from 219 infants and children. The kit was used according to the manufacturer's protocol on unextracted serum or plasma and also on reconstituted material extracted from the serum with propanol-heptane. The extraction protocol recovers approximately 88% of 17-OHP. Patients were grouped as infants 3-90 days (96 subjects) or older children, adolescents and young adults 91 days-20 years (123 subjects). 17-OHP results by the direct and extraction protocols correlated but the slopes of the regression lines (0.43 and 0.63) differed in the two groups, indicating that only about 49% of the immunoreactive material measured by the kit in the infants was 17-OHP whereas the corresponding percentage was 72% in the older children. Despite this nonspecificity, the present antibody is much more specific for 17-OHP in the presence of neonatal plasma steroids than that used previously. Reference values were obtained for the two groups using the method with and without an extraction step. 17-OHP values on four untreated patients with CAH were clearly elevated at the time of diagnosis. It is recommended that when the kit is used with neonatal and infant samples, an extraction step should be incorporated to enhance specificity.

Direct Enzyme-Immunoassay for Plasma Androstenedione

Abstract A competitive and sensitive enzyme-linked immunoadsorbent assay (ELISA) allowing determination of androstenedione (A) in unextracted human plasma is described. A highly specific antiserum (anti-4-androsten-11α-ol-3,17 dione-11α hemisuccinate-bovine serum albumin) was employed. Horseradish peroxidase (HRP) was used as the label enzyme; free-bound separation was carried out by adsorbing purified IgG of antiserum on polystyrene balls. Plasma protein binding capacity was reduced by adding 8-anilinonaphtalene-1-sulfonic acid ammonium salt (ANSA). After 3h-incubation, enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride as chromogen and hydrogen peroxide as substrate. The sensitivity of the assay was 5 pg/tube. In order to compare ELISA with radioimmunoassay (RIA) A estimations, plasma samples from thirty women with different endocrine disorders were assayed. A good correlation was found between the two techniques: r = 0.930, p. > 0.001. The accuracy, reprodu...

Development and validation of an immunoradiometric assay of parathyrin-related protein in unextracted plasma

This two-site immunoradiometric assay for human parathyrin-related protein 1-86 (PTHRP1-86) in plasma uses a mouse monoclonal antibody to PTHRP1-34 coupled to cellulose particles for immunoextraction of N-terminal immunoreactivity, and a rabbit antiserum to PTHRP37-67 that is indirectly labeled with 125I-labeled PTHRP37-67 for quantifying the bound analyte. The detection limit of the assay is 0.23 pmol/L, corresponding to 0.4 pg (0.04 fmol) per tube, for a sample volume of 200 microL. Recovery of PTHRP1-86 added to serum is essentially quantitative, and within- and between-batch precision is 4.4% and 11.1%, respectively. PTH1-84, PTHRP18-34, PTHRP9-34, PTHRP1-34, and PTHRP37-67 do not cross-react in the assay at concentrations up to 2 nmol/L. Plasma concentrations of PTHRP1-86 were below or close to the detection limit of the assay in normal subjects and in patients with primary hyperparathyroidism, hypoparathyroidism, chronic renal failure, and normocalcemic malignancy. In 37 hypercalcemic patients with various malignancies, we found detectable PTHRP1-86 concentrations in 35 (95%, mean 7.4 pmol/L, range 0.46-24.7). The data support the proposed humoral role of PTHRP in cancer-associated hypercalcemia and suggest that the assay has clinical utility in the differential diagnosis of hypercalcemia.

Pancreatic Release of Pancreastatin in the Pig

It is known that pancreastatin-like immunoreactivity (PLI) occurs in the secretory granules of the islet B- and D-cells in the pig pancreas, and that porcine pancreastatin inhibits insulin secretion in rats and mice. In this study, we characterized the porcine plasma PLI and examined whether PLI is released from the pig pancreas in vivo. We found that PLI in unextracted pig plasma largely consists of two high-molecular fractions, with Mr values of 80-85,000 and 300-350,000, respectively. In addition, a small peak of PLI eluted after gel filtration at the position of synthetic porcine pancreastatin. After extraction on octadecylsilyl silica, virtually all PLI disappeared except in the fraction co-eluting with porcine pancreastatin. In thiopenthal-anesthetized pigs, plasma samples were obtained from the carotid artery and the superior pancreaticoduodenal vein. By multiplying the venous-arterial concentration difference by the pancreatic venous plasma flow, a net pancreatic output of PLI of 420 +/- 120 pmol/min was found. This pancreatic PLI output was significantly reduced by electrical stimulation of the local autonomic nerves along the superior artery during atropine administration (p less than 0.001). Furthermore, the pancreatic venous PLI levels were elevated during intravenous infusion of glucose (p less than 0.01). We conclude that pig plasma PLI levels can be measured by radioimmunoassay after extraction on octadecylsilyl silica and that there is a net pancreatic output of PLI, which is reduced by sympathetic stimulation and enhanced during hyperglycemia.

Evaluation of a simple plasma catecholamine extraction procedure prior to high-performance liquid chromatography and electrochemical detection

The modified extraction method for catecholamines described in this study is reproducible, simple, rapid, economical and relatively hazard-free. This method is based on the principle that plasma catecholamines are selectively adsorbed on acid-washed alumina at pH 8.6 and then eluted at a pH between 1.0 and 2.0. No statistically significant differences were obtained by using either 0.5 or 1.0 ml of plasma with 0.5 or 1.0 ml of Tris buffer. A 15-min mixing time during the adsorption and desorption steps was found to be practical, but any standardized time up to 1 h can be used. If the washing step was omitted, the catecholamines could not be eluted from the acid-washed alumina. To prevent dilution, the alumina had to be centrifuged and not aspirated to dryness after the washing step. An amount of 50 mg of WA-4 alumina was found to be the most practical in this study. Extracted or unextracted plasma as well as catecholamine standards were stable for four months at -20 degrees C.

A Case of Dwarfism with Severely Reduced Activity of Growth Hormone-Binding Protein

We presented a 16-year-old boy with severe growth retardation and markedly decreased levels of growth hormone-binding protein (GHBP) in plasma, which was shown to correspond to the extracellular composition of hepatic GH receptor and suggested to reflect tissue concentration of the receptor. His height was 92.5 cm (-13.5 SD), the weight 9.6kg (-5.8 SD) and Tanner stage was I. His bone age was 3.5 years old at 16 years of age. Karyotype was 46,XY and thyroid function was normal. SM-C levels, determined by Nichols RIA using unextracted plasma, were within the low normal range, 0.67/0.68U/ml. In contrast, using a method of acid-ethanol extraction, IGF-I and IGF-II levels were definitely low, 29ng/ml (normal 88-240) and 165ng/ml (374-804) respectively. GH responses in various provocation tests, including insulin, arginine and GRF, were within normal. Basal GH levels were 20 +/- 12ng/ml and urinary GH excretion rates 217 +/- 85pg/mg. Cr, which were elevated compared to age-matched control. Molecular size of his circulating GH was similar to control subjects. The biological activities of GH, evaluated by radioreceptor assay and Nb2 cell bioassay, were proportional to the immunoactivities of GH. SM bioactivities, which were determined by the stimulatory effects on DNA synthesis of rabbit costal chondrocytes and human fibroblasts, were apparently reduced. Electrophoretic patterns of IGF-binding protein was similar to those of GH deficient cases. Daily administration of hGH (4U/day) for 5 days resulted in a poor response of SM-C production (before 0.68, after 0.77U/ml). GHBP activities were definitely low by gel-filtration, immunoprecipitation and charcoal methods, as seen in Laron dwarfism which is defined as a syndrome of congenital GH receptor defects. These results indicate that the tissue content of GH receptor in this case was quantitatively reduced and as a result, he showed a resistance to endogenous and exogenous GH. It remains to be elucidated whether the GH receptor defect in our case is derived from a genetic origin or an acquired condition.

Corticotrophin‐Releasing Factor‐41 in the Human and Rat—Utility of a Highly Sensitive Enzyme Amplified Immunometric Assay

Abstract We have developed a highly sensitive and specific immunoassay for human/rat corticotrophin-releasing factor-41 (CRF-41) to enable determination of immunoreactive CRF-41 levels in biological samples. To achieve high specificity, sensitivity and speed we have used two antisera in a sandwich enzyme immunoassay combined with enzyme amplification. The assay has a sensitivity of 0.08 fmol/well compared with radioimmunoassay sensitivities of 0.5 fmol/tube and is highly specific for the intact CRF-41 molecule. Measurement of samples is complete within 24 h compared with the 5 days required to obtain sensitive radioimmunoassay measurement. The assay has been used to measure both rat hypothalamic CRF-41 tissue content and release in vitro with good correlation when compared to radioimmunoassay measurement using antisera rC70 (0.983) or R1 (0.953). The assay only measures immunoreactive CRF-41 coeluting with human/rat CRF-41 and its oxidized form Met [O(21,38)]CRF-41 in human and rat tissue extracts separated by high-performance liquid chromatography. The ability to measure immunoreactive CRF-41 in unextracted plasma allows rapid measurement and eliminates multiple extraction steps.

Enzyme immunosorbant assay of oestradiol in unextracted plasma using penicillinase as label

An enzyme-linked immunosorbant assay (ELISA) for measuring oestradiol directly in plasma without extraction utilizing antibodies raised against oestradiol-3-( O-carboxymethyl) ether-bovine serum albumin conjugate, and oestradiol-6-( O-carboxymethyl) oxime linked to penicillinase (EC 3.5.2.6) as a marker was developed. Polyvinyl 96-well microtitre plates were used for immobilization of antioestradiol IgG. Standards of oestradiol (92 to 9190 pmol/l were prepared in oestradiol-free plasma and 8-anilino-l-naphthalene sulphonic acid (8-ANS, 5 mg/ml of 10 mmol/l PBS) was added to the microtitre plate wells to displace oestradiol from plasma binding proteins. The assay had a lower limit of detection of 92 pmol/l plasma and could be performed within 4 h. Comparison of oestradiol values of 51 plasma specimens obtained by ELISA with those of radioimmunoassay (RIA), in which oestradiol was extracted with diethyl ether, showed good correlation ( y = 0.786 x + 0.03; r = 0.900).

Immunoradiometric assay of atrial natriuretic peptide in unextracted plasma

We have developed and validated a two-site liquid-phase immunoradiometric assay (IRMA) of atrial natriuretic peptide (ANP) in unextracted human plasma. Both radiolabeled rabbit anti-ANP IgG and polyclonal mouse anti-ANP must bind to ANP for detection, and the assay is specific for peptides with both an intact C-terminus and a disulfide bridge. The assay sensitivity (detection limit) is 0.96 pmol/L, and the working range is 2.3-300 pmol/L, with the hook effect occurring above 500 pmol/L. Results for diluted plasma from normal subjects and from patients with renal failure paralleled the standard curve; analytical recovery of ANP added to such samples averaged 94%. The between- and within-assay CVs at 8 pmol/L were 10% and 5%, respectively. The assay is sufficiently sensitive and precise to detect the postural change in ANP concentrations in normal subjects.

Alteration of 5-HT uptake by plasma fractions in the premenstrual syndrome

The effects of plasma and an aqueous plasma fraction from patients with premenstrual syndrome (PMS) and control subjects on the uptake of 5-hydroxytryptamine (5-HT) in washed human platelets and rat forebrain synaptosomes were studied. Pre- and postmenstrual samples of unextracted plasma from the control group significantly enhanced platelet uptake of 5-HT. In contrast, an aqueous fraction following extraction of the plasma with organic solvents caused a dose-dependent decrease of 5-HT uptake. Plasma obtained from patients with PMS caused less stimulation of 5-HT uptake compared to plasma from the control group. The aqueous fraction of premenstrual plasma from patients tended to inhibit 5-HT uptake to a greater extent than a similar plasma fraction from controls. The inhibition of 5-HT uptake was associated with an increase in Km. Aqueous plasma fractions from both groups also inhibited 5-HT uptake in brain synaptosomes. However, there were no significant differences between groups. The results of the platelet study suggest that there may be quantitative differences in the plasma concentration of endogenous factors that affect 5-HT uptake between patients with PMS and control subjects and that such differences may explain the previously reported alteration of platelet 5-HT uptake and content associated with PMS symptoms.

Absence of Ultradian Rhythm or Diurnal Variation in Insulin-Like Growth Factor-I in Rats

We studied the variation in plasma insulin-like growth factor-I (IGF-I) concentrations in unrestrained, cannulated rats. To detect rapid changes, 6 rats were sampled every 15 min for 6 h. Plasma was assayed for growth hormone (GH), and for IGF-I before and after acid-ethanol (AE) extraction to reduce the masking effect of plasma binding proteins. AE increased the immunoreactivity of freshly collected rat serum and heparinized plasma by 3-fold, but had no effect on serum after 4 weeks of storage at -20 degrees C. In contrast, heparinized plasma maintained its sensitivity to AE for many months after collection. AE-extracted serum was identical in potency to serum that was subjected to acid gel chromatography. GH showed high amplitude, synchronized pulses every 3.3 +/- 0.15 h. Despite variations in IGF-I concentrations that resembled pulsations, when the data from both unextracted and AE-extracted plasma were subjected to 2 computer algorithms designed to detect hormone pulses, no pulses were identified in 8 of the 24 series of analyses. The two programs concurred only 4 times in their identification of a pulse in unextracted plasma, and in only one instance was a pulse identified in both unextracted and AE-extracted plasma from a given sample. Based on repeated measurements of a single serum sample, the program of Santen and Bardin (S&B) had a false-positive rate of 11%, and that of Merriam and Wachter (M&W) less than 10%. There was no positive correlation between summed GH and IGF-I concentrations over the 6 h of sampling, or after a 1- or 2-hour lag period. To detect diurnal variations, a second group of 5 rats was sampled every 2 h for 36 h. These animals showed little fluctuation of IGF-I concentrations and no differences between the light and dark periods. Our studies provide no evidence for episodic release of IGF-I or diurnal variations entrained to light or feeding cycles.

Advantages of IRMA over RIA in the Measurement of ACTH

A technically simple and rapid two-site immunoradiometric assay (IRMA) for human ACTH, based on monoclonal antibodies (MAbs), was compared with a clinically validated ACTH radioimmunoassay (RIA). Both methods measure ACTH 1-39 in unextracted plasma and cross-react less than 0.5% with ACTH fragments. ACTH levels were assessed in 103 patient samples: for concentrations in the range 5.3-1000 ng/L, results by the two methods were significantly correlated (r = 0.82, n = 86, P less than 0.001). The IRMA was more sensitive and had a wider working range than the RIA (detection limits 5.3 ng/L (IRMA) vs 11 ng/L (RIA); CV less than 10% between 19 and 1000 ng/L (IRMA) and CV less than 15% between 30 and 400 ng/L (RIA). In two patients for whom discrepant results were obtained, measurement of ACTH by bioassay and ACTH precursors by direct IRMA demonstrated the greater accuracy of the ACTH IRMA result. The improved performance of the IRMA combined with its many practical advantages compared to RIA, make it ideal for use in detailed clinical and physiological studies which have previously been hampered by the poor reliability of ACTH measurement.

Triton X-100 eliminates plasma proteins interference in a radioimmunoassay for luteinizing hormone-releasing hormone (LHRH) and LHRH analogues

A method is described for the radioimmunoassay of native LHRH and DTrp 6-LHRH, an LHRH analogue which does not require extraction of plasma samples. Interference by binding proteins normally present in plasma is removed by addition of Triton X-100 to the binding buffer at a concentration of 1% for LHRH and 0.15% for the LHRH analogue. This approach permits a direct estimation of the peptide level in unextracted plasma with quantitative recoveries for concentrations ranging from 0.015 to 10 ng/ml. Although the antiserum titre is reduced, the affinity of the antibody does not change at the detergent concentrations used in this study. This procedure is recommended for peptide assays in which the non-specific effects of plasma prevent a direct assay.

Development and validation of a two-site immunoradiometric assay for human atrial natriuretic factor in unextracted plasma.

This two-site immunoradiometric assay (IRMA) for human atrial natriuretic factor (ANF)99-126 in plasma utilizes a mouse monoclonal antibody raised against rat (r) ANF103-125 with specificity directed towards the ring structure of ANF, and a rabbit antiserum to human (h) ANF99-126. The monoclonal antibody is radioiodinated, and the IgG fraction of the antiserum is coated onto wells of a microtiter plate. Plasma or standard hANF99-126 (150 microL) is incubated with the radioligand in coated wells for 24 h. The detection limit is 0.9 pg per well, corresponding to 2 pmol/L, with a working range (CV less than 10%) from 4.5 to 540 pmol/L. Intra- and interassay precision are 7% and 9%, respectively, and the assay is unaffected by plasma matrix. In humans, the IRMA is specific for hANF99-126, the major circulating form of ANF, and does not cross-react with metabolites having deletions at the carboxy terminus. Plasma IRMA values in normal seated subjects were 6.6 +/- 1.5 (SEM) pmol/L and results correlated with those of an extraction RIA (r = 0.81, P less than 0.001).

Plasma concentrations of cholecystokinin, CCK-8, and CCK-33, 39 in rats, determined by a method based on enzyme digestion of gastrin before HPLC and RIA detection of CCK.

A new specific method for determination of cholecystokinin, CCK-8, and CCK-33, 39 in rat plasma is described. Plasma CCK radioimmunoassay (RIA) is difficult, because of cross-reactivity with gastrin. In the rat, problems because of difficulties in separating gastrin from CCK by high performance liquid chromatography (HPLC) exist. These were solved by enzyme digestion of gastrin before HPLC separation of molecular variants of CCK from gastrin fragments. Cholecystokinin immunoreactive forms in the HPLC fractions were determined by an antibody, which recognises the carboxyl terminus of CCK and gastrin. Fasting concentrations of small (CCK-8) and large (CCK-33, 39) molecular forms of CCK averaged 1.9 (0.3) pM and were raised to 13.4 (3.8) pM in rats fed ad libitum. Cholecystokinin in lactating rats rose two-fold after suckling, compared with 2.8 fold in response to feeding. The basal ratio between CCK-8 and CCK-33, 39 was approximately 1:1, but increased in favour of CCK-8 after feeding and in response to suckling. Gastrin like immunoreactivity measured in unextracted plasma was found to rise after feeding, but was unchanged in response to suckling.

Evaluation of a direct assay for atrial natriuretic peptide

A direct radioimmunoassay for the assessment of human atrial natriuretic peptide (ANP) in plasma, using a highly specific antibody and a well-defined monoiodotyrosyl-tracer was developed and evaluated by concurrent application of an extraction method. Sensitivity was 13.4 pg/ml; intra- and interassay variations were 3.1 and 5.5%, respectively; recovery averaged 99%; normal values ranged from 15–111 pg/ml (mean ± SD = 59 ± 25 pg/ml, n = 41). The results, including the effect of exercise, of the two methods correlated well. Pooling ANP values in normal subjects and patients with congestive heart failure gave a good correlation ( p < 0.01). However, due to processes in unextracted plasma, in the lower range the results from the direct method were erratic. Velocity and duration of centrifugation changed the number of platelets, but no effect on ANP levels, whether assessed by the direct or by the extraction method, was observed. Although the direct method is considerably less laborious than the extraction method its lack of reliability disqualifies it for most purposes.

Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay.

An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful.

The metatarsal cytochemical bioassay of parathyroid hormone: validation, specificity, and application to the study of pseudohypoparathyroidism type I.

The most sensitive method for assaying the bioactivity of PTH in unextracted plasma is the renal cytochemical bioassay. However, PTH acts on bone as well as kidney and clinical studies have suggested that the actions of circulating PTH level may be different at the two sites. We developed cytochemical bioassay for PTH based on the stimulation of glucose 6-phosphate dehydrogenase activity in the hypertrophic chondrocytes of the growth plate and the osteoblasts lining the metaphyseal trabeculae of rat metatarsal bones. The index of precision was 0.14 +/- 0.02 (SE) and the interassay variation was 31%. With this assay, plasma bioactive PTH levels in normal subjects and patients with primary hyperparathyroidism ranged from 0.5-18 ng/L and from 27-850 ng/L, respectively. Studies of patients with pseudohypoparathyroidism type I indicated that plasma PTH bioactivity in such patients is greater in the metatarsal bioassay than in the renal bioassay; no such differences were found in normal subjects or patients with primary hyperparathyroidism.

Ethanol increases and vitamin D metabolites decrease the production of cyclic AMP by canine renal cortical membranes.

Ethanol 0.16% increased cyclic AMP production by canine renal cortical membranes in the basal state and when challenged with different parathyroid hormone or fluoride concentrations. 1,25-dihydroxycholecalciferol (1,25(OH)2D3) 40 pM completely inhibited this effect of ethanol and reversed cyclic AMP production to the level observed in buffer alone. The same inhibitory effect was observed with 25OHD3 and with 24,25-dihydroxycholecalciferol (24,25(OH)2D3). The inhibitory effect was related to the vitamin D metabolites' concentration and was maximal for 160 pM; it was independent of their biological activity. This suggests that the effect is mediated through an interaction with the membrane lipids. The effect of vitamin D metabolites on cyclic AMP production was also observed in the presence of serum proteins and should be taken into account if unextracted plasma is assayed in the renal cortical membrane system for PTH bioactivity.