Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole

A method is described for increasing the response of enzyme immunoassays employing alkaline phosphatase as the label initiating 2 sequential catalytic reactions. First, NADP is dephosphorylated to produce NAD, which catalytically activates a specific redox-cycle involving the enzymes alcohol dehydrogenase and diaphorase. During each turn of the cycle 1 molecule of a tetrazolium salt is reduced to an intensely coloured formazan. The method is capable of detecting as little as 0.01 amol alkaline phosphatase, and when applied to an immunoassay for TSH a sensitivity (zero + 2.5 standard deviations) of 0.0013 mIU/l was obtained.     

A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry

A method for greatly enhancing the sensitivity of assays employing enzyme labels is described which offers advantages in assays for a wide range of analytes. The principle of the new approach is that the enzyme label gives rise to a catalytic activator for a specific secondary detection system, the activity of which is measured and related back to the amount of label present and thus of the analyte it is being used to determine (C.H. Self, Eur. Pat. Appl. 80303478.4, 15.4.81 exclusively licenced to IQ (Bio) Ltd.). The general principle of enzyme amplification is illustrated by using alkaline phosphatase as the labelling enzyme and nicotinamide adenine dinucleotide phosphate (NADP) as its substrate. The nicotinamide adenine dinucleotide (NAD) formed catalytically activates a strictly NAD specific redox cycle which produces a coloured formazan as the end product. The measured absorbance is at least two orders of magnitude greater than that achieved by conventional methods. The application of this method to immunoassay is demonstrated by a sensitive, rapid and precise assay for human prostatic acid phosphatase (PAP). Some of the many other applications of this methodology are discussed.     

The effects of silver on intestinal ion and acid-base regulation in the marine teleost fish, Parophrys vetulus

Exposure to elevated silver (as AgNO3) concentrations (6-9 microM) in seawater was associated with comparably high silver concentrations in the intestinal fluids of the lemon sole (Parophrys vetulus), and a tendency for reduced drinking rate. The effects of silver on intestinal ion and acid-base regulation were studied using in situ perfusion of the intestine. Intestinal net Cl- uptake was reduced from 0.4 to 0.1 and intestinal net Na+ uptake from 0.2 to 0 mmol kg(-1) x h(-1) during silver exposure (9 microM). At the same time, intestinal HCO3- net efflux was reduced from 0.2 to 0.1 mmol kg(-1) x h(-1). Both intestinal Na+ and Cl- uptake and Cl-/HCO3- exchange are thus sensitive to silver, but to different extents. None of the observed effects were reversible during 24 h of recovery. Intestinal water transport was highly variable in vivo in the perfused preparation, and no significant effect of silver exposure was observed. However, in vitro intestine preparations exhibited reduction of intestinal net water flux from 4 to 1 microl cm(-2) x h(-1) during silver exposure together with reduced unidirectional Cl- influx. Reduced water intake and transepithelial water transport in silver-exposed fish resulted in moderate hemoconcentration evident from higher hematocrit values, but not in increased plasma ion levels. The latter could reflect a compensatory response via increased branchial Na+/K+-ATPase levels, observed in silver-exposed fish, indicative of increased branchial ion transport capacity. Impairment of intestinal ion and water transport as a result of silver intake via drinking could be an important part of the fatal cascade of physiological effects observed in marine fish during acute silver exposure.     

Comparison of the metabolic effects of raloxifene and oral estrogen in postmenopausal and growth hormone-deficient women

CONTEXT: The endocrine and metabolic functions of the liver are affected by estrogen. Oral estrogen reduces IGF-I and suppresses fat oxidation despite augmenting GH secretion. The aim of this study was to determine whether selective estrogen receptor modulators display similar effects and whether these effects are magnified in GH-deficient (GHD) women because of the loss of GH feedback. DESIGN: This was an open-label, randomized, two-period, crossover study comparing treatment (raloxifene vs. estradiol) and group (normal vs. GHD). SETTING: The setting of this study was a clinical research unit. PARTICIPANTS: Twelve postmenopausal women and 12 women with hypopituitarism participated in this study. INTERVENTION: Two 4-wk treatments with 17beta-estradiol (E2; 2 mg, followed by 4 mg) or raloxifene (60 mg, followed by 120 mg) were given, crossing over to the alternate treatment after a 4-wk washout period. OUTCOME MEASURES: Endocrine [GH, IGF-I, IGF-binding protein-3 (IGFBP-3), GH-binding protein, and SHBG] and metabolic (fat oxidation) end points were used as outcome measures. RESULTS: E2 reduced serum IGF-I levels in a dose-dependent manner in both groups, with effects greater (P < 0.05) than raloxifene. Raloxifene reduced IGF-I levels in the GHD group (P < 0.001), but not in the postmenopausal group. E2 reduced (P < 0.05), and raloxifene increased (P < 0.05), IGFBP-3 levels in both groups. E2, but not raloxifene, increased GH (P < 0.05) in postmenopausal women. The effects of E2 and raloxifene on IGF-I, IGFBP-3, IGF-I/IGFBP-3 molar ratio, GH-binding protein, and SHBG were significantly different (P < 0.05). E2 and raloxifene reduced (P < 0.05) fat oxidation equally in GHD, whereas the decrease in postmenopausal women was not significant. CONCLUSION: E2 and raloxifene exert different hepatic endocrine, but not lipid oxidative, effects. The greater effects seen in GHD women may be explained by the loss of endogenous GH feedback.     

The reduction in visceral fat mass in response to growth hormone is more marked in men than in oestrogen-deficient women

ContextWomen with severe growth hormone (GH) deficiency have a less marked response to GH replacement than men. This has mostly been attributed to the attenuating effects of oestrogen replacement therapy.ObjectiveTo study gender related differences in the response to GH treatment in men and postmenopausal women.MethodsFifteen men and 15 age- and BMI-matched women with abdominal obesity (mean age: 58; range 51–64 years) were treated for one year with similar doses (0.47 vs. 0.51 mg/day) of GH. All women were postmenopausal not receiving oestrogen treatment. Insulin sensitivity was assessed using a hyperinsulinemic euglycemic clamp and body composition by computed tomography (CT) scans and from total body potassium, K⁴⁰.ResultsMen and women were comparable at baseline in terms of waist circumference, IGF-1 and lipid levels. After one year of GH treatment, there was a 18% reduction in visceral adipose tissue (VAT) in men and a 5% reduction in women (P = 0.0001 men vs. women). Although the magnitude of the difference was small, men increased more in thigh muscle mass (P < 0.0001 vs. women). A reduction in thigh intermuscular adipose tissue (IMAT) and diastolic blood pressure was seen only in men (both p < 0.05 vs. baseline). A decrease in LDL cholesterol, and an increase in serum insulin, was observed only in women (both p < 0.05 vs. baseline).ConclusionLow dose GH treatment reduced VAT more markedly in men as compared with women. As all women were postmenopausal and oestrogen-deficient, this gender difference in responsiveness was not due to an antagonistic effect of oestrogen on peripheral GH action.     

Baseline characteristics and the effects of five years of GH replacement therapy in adults with GH deficiency of childhood or adulthood onset: a comparative, prospective study

The consequences of GH deficiency may differ if the disease is childhood onset or adulthood onset. In this single-center, prospective study, 21 consecutive adults with childhood onset GH deficiency and 21 adults with adulthood onset GH deficiency, matched for age, gender, body mass index, and number of anterior pituitary hormonal deficiencies, were included. Baseline differences and differences in the responses in body composition, muscle strength, bone mass, and metabolic indices during 5-yr GH replacement were determined. The duration of GH deficiency was longer and serum IGF-I level and body height were lower in the childhood onset patients than in the adulthood onset patients. Body fat (observed/predicted ratio) was increased, and lean mass and muscle strength were decreased, in the childhood onset patients. Total body and lumbar (L2-L4) bone mineral content and bone mineral density were lower in the childhood onset patients. Serum total cholesterol level was higher in the adulthood onset patients. The childhood onset and adulthood onset patients received a similar dose of GH. After adjustment for body weight, however, the dose of GH was higher in the childhood onset patients. The treatment responses were more marked in the childhood onset patients in lean mass, knee extensor strength, left-hand grip strength, and in total body and lumbar (L2-L4) bone mineral content and bone mineral density. The reduction in serum total cholesterol concentration was more marked in the adulthood onset patients. At study end, no differences remained between the two study groups after the correction for body height in the statistical analysis. In conclusion, the baseline analysis suggests more decreased lean mass, muscle strength, and bone mass in the childhood onset patients whereas the lipid profile was more disturbed in the adulthood onset patients. The 5-yr GH replacement eliminated all the anthropodometric and metabolic differences between the two groups.     

Baseline characteristics and the effects of two years of growth hormone (GH) replacement therapy in adults with GH deficiency previously treated for acromegaly

CONTEXT: The effects of GH replacement in GH-deficient (GHD) adults previously treated for acromegaly are not well known. OBJECTIVE, DESIGN, AND PATIENTS: In this single-center, open-labeled, prospective study, 10 consecutive GHD adults with cured acromegaly (A group) and 10 matched GHD adults with previous nonfunctioning hypopituitary disease (NF group) were included. Comparisons were made at baseline and in the responses in body composition, muscle strength, bone mass, and metabolic indices during 2 yr of GH replacement. RESULTS: At baseline, upper leg local muscle endurance and serum low-density lipoprotein-cholesterol concentration were more impaired in the A group. The A group contained three patients with hypertension, one with diabetes mellitus type 2, and one with hyperlipidemia. The NF group had only one patient with hypertension. There were no significant between-group differences in the responses to the GH therapy. Body composition and serum lipid pattern improved in both groups without any deterioration of glucose homeostasis. At study end, no difference remained between the two groups in any variable. During the 2-yr treatment, one patient had a myocardial infarction and two had cerebral infarctions in the A group, whereas no vascular event occurred in the NF group. CONCLUSIONS: GHD patients with previous acromegaly have an impaired cardiovascular risk profile and decreased local muscle endurance as compared with other GHD patients. Two-year GH replacement eliminated these differences, but vascular events occurred more frequently in the A group. Therefore, GHD patients with cured acromegaly will benefit from GH replacement, but careful monitoring of cardiovascular status is needed.