Insulin-like growth factor binding protein-3 generation as a measure of GH sensitivity.

A total of 198 subjects were randomized to either high-dose (0.05 mg/kg.d) or low-dose (0.025 mg/kg.d) GH for 7 d; the alternate dose was then received after a 2-wk washout period. Groups included in the study were: normal, GH-insensitive (GHI; homozygous for the E180 splice mutation); heterozygous GHI (carriers of the E180 splice mutation); GH-deficient; and idiopathic short stature. Serum IGF binding protein-3 (IGFBP-3) concentrations (collected on d 1, 5, and 8 of treatment weeks) were GH-dependent, with significant elevation by d 5 of treatment, regardless of dose, in all normal subjects. GHI subjects had low baseline IGFBP-3 and poor or no response to either low- or high-dose GH. Heterozygous subjects, however, did not differ from age-matched normals with regard to IGFBP-3 generation. All GH-deficient subjects had subnormal baseline concentrations of IGFBP-3; most, but not all, were able to generate levels into normal ranges by 8 d of therapy. Children with idiopathic short stature showed a better response in IGFBP-3 generation compared with that previously observed with IGF-I, reaching concentrations in normal range with either dose of GH, suggesting that any GHI in this group is relatively limited to IGF-I production. For the diagnosis of GHI, the highest sensitivity (100%) and specificity (92%) was found on d 8 of the high-dose GH-IGFBP-3 generation test. Failure to raise both IGF-I and IGFBP-3 lowered sensitivity to 82-86% with low-dose GH, and 86-91% with high-dose GH.

The IGF-I generation test revisited: a marker of GH sensitivity.

IGF-I generation tests were developed over 20 yr ago and are currently used in differentiating GH insensitivity (GHI) from other disorders characterized by low serum IGF-I. Nevertheless, generation tests have never been adequately characterized, and insufficient normative data are available. One hundred and ninety-eight subjects [including normal subjects; subjects with GHI, GH deficiency (GHD), and idiopathic short stature (ISS); and heterozygotes for the E180 splice GH receptor mutation] were randomized to self-administration of either a high (0.05 mg/kg x d) or a low (0.025 mg/kg x d) dose of GH for 7 d. After a 2-wk washout period, they received the alternate dose. Samples were collected on d 1, 5, and 8 of each treatment period. In normal individuals, IGF-I generation was GH dependent at all ages, and little advantage was observed in using the higher dose of GH or extending beyond the d 5 sample. Some GHD patients had IGF-I levels, both baseline and stimulated, that overlapped levels in the verified GHI patients. Subjects heterozygous for the E180 GH receptor splice mutation did not show a decreased responsiveness to GH. ISS patients had low-normal IGF-I levels that did not stimulate beyond the baseline normative ranges for age. These data provide the first large scale effort to provide preliminary normative IGF generation data and evaluate the GH sensitivity of patients with GHI, GHD, and ISS.

Usefulness of different biochemical markers of the insulin-like growth factor (IGF) family in diagnosing growth hormone excess and deficiency in adults.

The diagnostic approach to acromegaly and GH deficiency frequently includes measurement of several components of the insulin-like growth factor (IGF) system. IGF-I levels are reported to be good predictors of active and cured acromegaly, but are commonly found within the normal age-adjusted range in adult GH-deficient (GHD) patients. Circulating concentrations of IGF-binding protein-3 (IGFBP-3), acid-labile subunit (ALS), and free IGF-I reflect the GH secretory status, but their diagnostic accuracy is still debated. In this study serum levels of total and free IGF-I, IGFBP-3, ALS, and IGFBP-3-IGF-I and IGFBP-3-ALS complexes were determined in patients previously diagnosed with active (n = 67) or inactive (n = 16) acromegaly and adult GHD (n = 34) and compared with results obtained in 58 healthy controls. In healthy subjects, IGF-I, IGFBP-3, ALS, and both IGFBP-3 complexes declined with age; a correlation was found between IGF-I and IGFBP-3 (r = 0.59; P < 0.001), ALS (r = 0.67; P < 0.001), and free IGF-I (r = 0.40; P < 0.05). Active acromegalic patients showed a significant increase in all parameters tested. IGF-I concentrations were above +2 SD in 100% of patients, whereas slightly lower sensitivities were shown for IGFBP-3 (85%), ALS (88%), and free IGF-I (94%). In this group, IGF-I exhibited a slightly higher correlation with IGFBP-3 (r = 0.83; P < 0.001) than with ALS levels (r = 0.78; P < 0.001). In cured acromegalic patients, we observed the normalization of all parameters but free IGF-I levels. Adult GHD patients showed a significant reduction of all hormones. Unlike active acromegalic patients, all parameters had only a modest sensitivity in GHD; suppression below -2 SD was observed in 41% of GHD patients for IGF-I, 47% for IGFBP-3, 32% for ALS, and 35% for free IGF-I measurements. Previous radiotherapy and GH peak response below 3 microg/L were associated with significantly lower IGF-I, IGFBP-3, and ALS levels. IGF-I levels were significantly correlated to ALS (r = 0.68; P < 0.001) and IGFBP-3 (r = 0.64; P < 0.001) as well as with free IGF-I (r = 0.67; P < 0.001) levels. By multiple regression analysis, the number of anterior pituitary hormones impaired was the most predictive indicator of IGF-I, IGFBP-3, and free IGF-I levels in GHD patients; conversely, the GH peak response better anticipated ALS concentrations. The pattern of IGFBP-3 complexes paralleled previous hormonal findings. In active acromegalic patients, IGFBP-3-IGF-I levels were 5.4-fold higher than in controls and were above +2 SD in 95% of patients, whereas IGFBP-3-ALS levels were elevated in 15% of cases. On the other hand, both IGFBP-3 complexes were able to predict GHD in only a minority of cases. Taken together, these data support the diagnostic role of IGF-I in acromegaly and suggest that free IGF-I and the IGFBP-3-IGF-I complex can assist diagnostic strategies in this condition. All markers are of limited predictive value in adult GHD, as hormonal values are commonly found within the normal limits. In these patients, low IGFBP-3 and IGF-I concentrations can add further clinical information on the residual GH activity.

Dithiothreitol treatment permits measurement of melatonin in otherwise unusable saliva samples.

Melatonin research has primarily utilized blood as the source of samples, but there is now increasing interest in measuring levels of the hormone found in saliva. One impediment to this approach is that several melatonin assays involve a column-extraction step that can prove very time-consuming or even impossible when salivary samples are excessively viscous. We have treated 67 samples with dithiothreitol to enhance their passage through the column. Following this treatment, all samples passed freely through the columns. The minimum and maximum values measured were 0.7 - 50.0 pg/ml for the untreated controls and 1.0 - 51.9 pg/ml for the treated samples. The means (+/-SEM) for these groups were 9.5 +/- 1.6 and 9.9 +/- 1.7, respectively, and were not significantly different from one another as assessed by Student's t-test (P = 0.08). In summary, we have found that this technique permits us to obtain values on samples which would otherwise be unusable and that such treatment does not alter the melatonin values yielded by RIA analysis.

Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils.

The subcellular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotaurine and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50:1. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiring cells by exposure to opsonized zymosan. These results prompted in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypotaurine competes with 5,5'-dimethyl-1-pyrroline N-oxide) (DMPO) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.

Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils.

Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.

Purification of the solubilized NADPH:O2 oxidoreductase of human neutrophils. Isolation of its catalytically inactive cytochrome b and flavoprotein redox centers.

The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.

A reassessment of the product specificity of the NADPH:O2 oxidoreductase of human neutrophils.

Native ferricytochrome c, but not acetylated ferricytochrome c, stimulates the flow of electron equivalents passing through the neutrophil NADPH:O2 oxidoreductase complex. At 28 mM it increases NADPH oxidase activity by 157 +/- 15% (n = 5) over that measured in its absence. Enhanced activity is predominantly seen in oxidoreductase-rich 27,000 X g membrane preparations obtained from phorbol myristate acetate activated cells. Superoxide formation is also enhanced. Although some of the stimulatory activity seen with addition of native ferricytochrome c to oxidoreductase-rich membrane suspensions might have been explained in terms of mitochondrial contamination, this was ruled out. Comparable membrane preparations from resting cells were devoid of NADPH oxidase activity. Azide, a well-known inhibitor of the electron transport chain, did not block the enhancing effect of native ferricytochrome c. These results indicate that native ferricytochrome c is not a suitable scavenger of superoxide in quantitating the product specificity of the oxidoreductase since it amplifies the apparent rate of superoxide formation with respect to measured rates of NADPH oxidation conducted in its absence. By using acetylated ferricytochrome c in place of native ferricytochrome c in quantitating the product specificity of the oxidoreductase we show that no more than 70% of the electron equivalents donated by NADPH to the oxidoreductase are involved in superoxide formation. The remaining 30% of the electron equivalents given up by NADPH to the oxidoreductase appear to be involved in direct formation of hydrogen peroxide.

Human lymphocytes can generate thymus-independent as well as thymus-dependent anti-hapten plaque-forming cell responses in vitro.

Human lymphocytes from peripheral blood, tonsils, and adenoids produced hapten-specific anti-TNP antibodies after in vitro stimulation with TNP-Ba. This antigen did not appear to behave as a polyclonal activator since TNP-Ba-stimulated cells lysed TNP-conjugated sheep erythrocytes (SRBC) but not unconjugated SRBC or DPPC-conjugated SRBC, whereas cells from PWM-stimulated cultures formed hemolytic plaques with both haptenated and unhaptenated targets. In addition, TNP-Ba generated PFC were inhibitable by soluble hapten (TNP-EACA or TNP-BSA). B cells depleted of T cells (less than 1% E-RFC cells) were able to respond to TNP-Ba but not to PWM or TNP-KLH. When varying ratios of T and B cells were added to cultures the TNP-Ba anti-TNP response was found to have a linear correlation with the number of B cells added (r = 0.987) and was maximal in the absence of added T cells. This was in contrast to the response to TNP-KLH, which required T cells at a particular T:B ratio for optimal induction of anti-TNP PFC. Thus, both T-dependent and T-independent anti-hapten responses could be elicited in human lymphoid cell cultures.

Techniques of EEG frequency analysis for evaluation of uremic encephalopathy.

In an effort to provide nephrologists with practical, objective, and quantitative methods for evaluating uremic encephalopathy and the severity of uremia, we investigated five techniques for EEG frequency analysis, the selection of EEG samples for analysis, and the normal values for dominant frequency and the percent of EEG power from one through six Hz. The five techniques consisted of 1. handcounting, 2. use of a tape recorder and sonic analysis system to determine EEG power versus frequency, 3. use of the tape recorder and sonic analysis system to determine EEG voltage versus frequency, 4. on-line measurement of % EEG voltage from one through six Hz, and 5. use of base line crossovers to count the number of waves occurring at each frequency. All techniques were found to be satisfactory. The most significant difference between techniques depended on whether wave amplitude influenced the analysis (techniques 2, 3, and 4) or did not (techniques 1 and 5); in the former case slow wave activity was more evident than in the latter case. Our experience indicated that the determination of relative EEG power from each frequency (EEG power versus frequency or power spectral density) was the most practical and revealing mode of analysis, but any of these techniques would be valid and useful if employed with awareness of the difference in the normal range obtained by different techniques.