Circulating nucleic acids in the assessment of endogenous growth hormone production.

There is growing concern about the use of recombinant human growth hormone (rhGH) by individuals taking part in competitive sports. Although rhGH is banned by the international organizations, the detection of GH doping is difficult. We postulated that rhGH will suppress endogenous GH production, which can be assessed by the measurement of mRNA for GH and growth hormone-releasing hormone (GHRH). In order to prove this concept, we undertook a pilot study to examine whether circulating nucleic acids are useful in the detection of endogenous GH production. Blood samples were collected into PAXgene tubes from 37 healthy controls and 12 acromegalic patients. RNA was extracted from the samples, cDNA was obtained, and the quantities of mRNA for GH and GHRH were measured using real-time PCR. In acromegalic patients, median mRNA concentration for GHRH (corrected for beta-actin mRNA) was 30.7 times lower than in controls (median delta C(T)) value of -0.128 versus 3.927, P < 0.001). There was a significant correlation between serum IGF-1 SD score and mRNA for GHRH (r= 0.407). In acromegalic patients, mRNA for GH was significantly higher than in controls (median values of -4.694 versus -0.044, P < 0.05). As GH production is known to decline with age, we also examined mRNA for GH and GHRH according to age subgroups. Both markers were significantly lower in the older age group (>50 years) compared to the younger age group (<34 years). These results show that mRNA for GH and GHRH can be detected in the peripheral circulation and raises the possibility of using these markers in the detection of exogenously administered GH.

Stability of beta-actin mRNA in plasma.

It has long been thought that mRNA is labile and easily prone to degradation. However, a recent study demonstrated that GAPDH mRNA in cell-free plasma may remain stable up to 24 hours after blood collection. As there are no other independent studies, we attempted to reproduce the findings of that study. In our study, blood was collected from a healthy male volunteer into Vacutainer tubes containing EDTA. Blood samples were placed on ice and plasma separated by double-centrifugation at times 0, 1, 2, and 5 hours after blood collection. mRNA was extracted from four aliquots of the blood sample by means of the QIAamp Viral RNA kit. Extracted mRNA was converted to cDNA by reverse transcription before real time quantitative PCR measurement of the housekeeping beta-actin gene. Plasma beta-actin mRNA at 2 hours (0.012; 0.0031-0.0297, median and range) was significantly lower (P= 0.022) than at 0 hours (0.12; 0.057-0.165) (P= 0.016). The levels decreased further at 5 hours (0.0037; 0.0024-0.011) (P= 0.004). The results show that plasma beta-actin mRNA levels decrease with time after blood collection and that this is likely to be due to degradation in vitro.

Normalization of circulating nucleic acid results.

Nucleic acids, both DNA and mRNA, have been detected in the circulation and have been demonstrated to be useful in such areas as fetal medicine, oncology, and transplantation. When mRNA is measured in circulating blood, the results are expressed in relation to a reference gene product in order to correct for any differences in extraction, volume of starting material or other differences. Many authors use beta-actin mRNA and express results as a ratio of target mRNA to beta-actin mRNA. We have used a similar approach when studying diabetic retinopathy. Recently, we planned to investigate the expression of thyroid dependent gene expression in acutely ill patients. As a control study, we examined the expression of thyroid hormone-dependent gene expression in subjects with hyperthyroidism and found that the expression of beta-actin mRNA was affected by thyroid hormone status. Blood samples were taken into PAX genetrade mark tubes from 31 healthy subjects (mean age, 43 +/- 16 yrs) and 7 patients with hyperthyroidism (mean age, 43 +/- 5 yrs). Diagnosis of hyperthyroidism was confirmed by clinical findings and biochemical results. After extraction of mRNA, cDNA was synthesized using reverse transcription. Quantification of Na/K-ATPase, T3 receptor, and beta-actin cDNA was carried out by RT-PCR. Median beta-actin levels were significantly higher in hyperthyroid subjects compared to healthy subjects (18.2 versus 2.30; P < 0.00042). When mRNA for the T3 receptor was expressed in relation to beta-actin, there was a significantly higher in hyperthyroidism (0.0168 versus 0.218, P < 0.05). However, this was significantly lower when expressed in relation to total RNA (12.2 versus 2.24, P < 0.00015). We conclude that normalizing results to beta-actin may not be appropriate in all circumstances.

Measurement of circulating neuron-specific enolase mRNA in diabetes mellitus.

In this study we measured the levels of neuron-specific enolase mRNA as a possible marker of diabetic neuropathy. Blood samples were collected from healthy controls (n= 26), diabetic controls (no known neuropathy or retinopathy) (n= 22), and diabetics with clinically diagnosed neuropathy (n= 24) into PAXgene blood RNA tubes. mRNA was extracted, reverse-transcribed to cDNA, and measured by real-time quantitative PCR. Enolase mRNA levels were normalized by the simultaneous measurement of beta-actin mRNA. The results showed that the enolase mRNA was significantly (P= 0.002) higher in the diabetic control (median = 0.018; range = 0.006-0.037) group compared to healthy subjects (median = 0.0086; range = 0.0016-0.039). However, the diabetic neuropathy group showed lower enolase levels (median = 0.0067; range = 0.0025-0.017) compared to both healthy subjects (P= 0.06) and diabetic controls (P < 0.001). In the diabetic neuropathy group patients with preproliferative (median = 0.01; range = 0.008-0.017) or proliferative retinopathy (median = 0.011; range = 0.007-0.015) had significantly (P= 0.001) higher enolase mRNA compared to patients with background retinopathy (media = 0.004; range = 0.0025-0.0092). It is concluded that levels of enolase mRNA are decreased in diabetic neuropathy and this molecular marker may also be useful in differentiating early from advanced eye disease in those diabetics diagnosed with neuropathy.

Retina-specific mRNA in the assessment of diabetic retinopathy.

In a previous study we demonstrated the presence and diagnostic usefulness of circulating rhodopsin mRNA in the assessment of diabetic retinopathy (DR). In the present study we investigated three further retina-specific markers in blood to determine their suitability as markers of DR. The markers were RPE65, retinoschisin, and melanopsin. Whole blood was collected from diabetic patients and healthy controls into PAXgene Blood RNA tubes and RNA was extracted using the PAXgene Blood RNA System. Quantitative real-time PCR was used to quantify mRNA for RPE65, retinoschisin, and melanopsin. beta-actin mRNA was used for normalization. RPE65, retinoschisin, and beta-actin mRNA were detected in 100% of subjects; melanopsin was not detected in either controls or diabetic patients. Circulating RPE65 mRNA concentration was 63% higher in diabetic patients than in healthy individuals (P= 0.019), whereas retinoschisin showed no change between the two groups. Compared with healthy controls, circulating RPE65 mRNA concentration was higher in diabetics with no retinopathy (30%; P= NS), background DR (93%; P= 0.01), preproliferative DR (20%; P= NS), and proliferative DR (107%; P= 0.004). Compared with diabetics with no retinopathy, levels of RPE65 mRNA were also significantly higher (60%) in the presence of proliferative DR (P= 0.029). In contrast, levels of retinoschisin mRNA were lower in background DR (34%; P= 0.033), preproliferative DR (43%; P= 0.026), and proliferative DR (47%; P= 0.038) compared to that in diabetics without retinopathy. We conclude that not all retina-specific mRNA species are detectable in circulation (e.g., melanopsin). This may be related to differences in expression levels for the individual markers. Both RPE65 and retinoschisin were detectable and demonstrated contrasting trends in diabetics with and without retinopathy. In combination with rhodopsin, RPE65, and retinoschisin, mRNA may offer a useful tool in developing a blood test for DR.

Hypertension and circulating mRNA for 11beta-hydroxysteroid dehydrogenase type II.

Circulating DNA and mRNA for 11beta-hydoxysteroid dehydrogenase (HSD) type II were measured in patients with hypertension and in healthy subjects. DNA and RNA levels in hypertensive patients and controls were quantified using real-time RT-PCR. Messenger RNA for 11beta-HSD type II was significantly lower in the hypertensive patients (median: 0.18) (P= 0.032) than in healthy subjects (median: 0.42). Plasma DNA was also significantly lower (P= 0.016) in hypertension. It is suggested that measurement of mRNA for 11beta-HSD type II in hypertension may identify subjects who may be salt-sensitive.

Binding of ouabain and human ouabainlike substance to different Na+, K+-ATPase isoforms.

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual alpha-isoforms of Na+,K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney alpha1, rabbit kidney alpha1 and porcine cerebral cortex alpha3 Na+,K+-ATPase. OLS was initially isolated by solid phase extraction from human serum using C18 columns. The extract was further purified by reverse phase HPLC in an acetonitrile/water (containing 0.1% TFA) step-up gradient (16-80%). In this system, two distinct ouabain immunoreactive peaks were resolved. Peak I demonstrated a polarity identical with that of authentic ouabain. In contrast, peak II was relatively non-polar and eluted later in the run. The final step in the purification of OLS involved immuno-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100 nmol/l) for ouabain with canine alpha1 and porcine alpha3 Na+,K+-ATPase showed similar inhibitory profiles (IC50=15 nmol/l), whilst rabbit alpha1 Na+,K+-ATPase was relatively insensitive to ouabain and purified peak I OLS. Two fold serial dilution of Peak I OLS, with subsequent analysis by canine and porcine Na+,K+-ATPase inhibition assays and RIA, demonstrated strong positive correlations between OLS determined by RIA and both canine (y=0.945x-2.532, r2=0.977) and porcine (y=0.428x-1.685; r2=0.993) Na+,K+-ATPase assays. The difference in the respective slopes suggests, however, that peak I OLS has a greater affinity for the canine derived enzyme compared to the porcine. In conclusion, these data suggest that like authentic ouabain, peak I OLS is a-isoform and species selective.

Endogenous ouabain secretion in man is not regulated by ACTH.

It has been suggested that endogenous ouabain-like substance (OLS) is of adrenal origin and the secretion of OLS may be ACTH dependent. To determine if OLS is influenced by the pituitary-adrenal axis, we studied the effect of adrenal stimulation (0.25 mg Synacthen) and suppression (1 mg Dexamethasone) on two separate groups of nine subjects. Serum OLS was measured by a radioimmunoassay (RIA) developed in our lab, and cortisol and ACTH were measured by commercial assay kits. Dexamethasone significantly (P< 0.001) suppressed serum cortisol and ACTH concentrations, without effecting endogenous OLS concentration (0.64+/-0.17 vs 0.85+/-0.18nmol/l). Synacthen increased the concentration of cortisol in serum (p < 0.001) over the test period; OLS concentration, again, remained unchanged (0.45+/-0.04 vs 0.43+/-0.05 nmol/l). In further studies, serum concentrations of cortisol and OLS were compared between left (LAV) and right (RAV) adrenal veins with that from the inferior vena cava (IVC). Concentration of cortisol in the LAV and RAV was five-fold greater than that in IVC. However, there was no difference in OLS concentration at the corresponding sites. In addition, serum OLS concentrations in patients having undergone bilateral adrenalectomy or diagnosed with Addison's disease (0.62+/-0.19 nmol/l) were similar to concentrations in healthy subjects (0.67+/-0.21 nmol/l). Examination of bovine adrenal, liver, kidney, heart and human placenta demonstrated that OLS content of bovine adrenal was comparable with other tissues analysed. HPLC studies of human serum and bovine adrenal gland produced identical elution profiles, resolving a single peak which coincided with the retention time observed for standard ouabain. We conclude that the adrenal is unlikely to be the source of endogenous OLS, the secretion of which appears to be independent of ACTH.

Effect of high salt intake on plasma and tissue concentration of endogenous ouabain-like substance in the rat.

The effect of high salt intake on serum concentration and tissue distribution of ouabain-like substance (OLS) was examined in rats. Sprague-Dawley rats (n=8) were placed on a high salt diet by the inclusion of 1.8% sodium chloride in drinking water for 7 days and a 'control' group (n=8) was maintained on normal drinking water during the study period. Serum and tissue OLS was measured by radioimmunoassay after solid phase extraction. High salt intake significantly increased serum OLS concentration (1.43 +/- 0.06 vs 1.14 +/- 0.05 nmol/L; mean +/- SEM, P=0.002). In both groups, the adrenal showed significantly (p < 0.001) higher OLS content compared to liver, kidney, heart and brain. HPLC of rat serum extract resolved a major peak with a retention time identical to that of standard ouabain, further confirming the nature of OLS. We conclude that high salt intake increases endogenous production of OLS, which appears to originate from the adrenal gland in the rat.

Effect of salt intake on excretion of endogenous ouabain-like substance, measured by RIA.

Recent studies suggest that ouabain or a ouabain-like substance (OLS) may be present endogenously in humans. We developed a RIA for ouabain with antisera raised in goat against ouabain conjugated to keyhole limpet hemocyanin and ovalbumin. The antiserum was of high antibody titer (200,000) and was specific for ouabain, with little cross-reactivity with common steroids and structurally related compounds such as ouabagenin (4%), strophanthidin (4%), and dihydroouabain (2%). The RIA had a working range of 0.06-2.0 nmol/L, and the intra- and interassay CV was 6.5% at a concentration of 1.7 nmol/L. With this assay the effect of salt loading on urinary excretion of OLS was examined in 10 healthy volunteers (ages 18-22 years) who increased their salt intake (sodium) for 5 days and reduced it for the next 5 days. Urine was collected and OLS concentration was measured by RIA after solid-phase extraction with a Bond Elut C18 column. Excretion of OLS and sodium were maximal on day 5 and lowest on days 9 and 10. Urine excretion of OLS on day 5 (2.66 +/- 1.22 nmol/24 h) was significantly higher (P < 0.0001) than on day 10 (1.47 +/- 0.69 nmol/24 h). We conclude that (a) the assay developed has sufficient sensitivity and specificity to detect endogenous OLS present in biological fluids, and (b) salt intake increases the excretion of OLS.

Cholesterol absorption by the gall bladder.

Model and real biles were used to investigate factors influencing cholesterol and dextran (70,000 molecular weight) absorption by the gall bladder. Cholesterol absorption was proportional to cholesterol concentration when real bile was used, but model biles showed maximal absorption at cholesterol saturation. Reduction of temperature reduced cholesterol absorption and serosal secretion, but had little effect on dextran absorption. This indicates differences in uptake where cholesterol undergoes passive diffusion but dextran is taken up by fluid-phase endocytosis. Model bile prepared with a single bile salt showed lowest cholesterol uptake from cholate bile, but there was no difference in serosal secretion. Dextran uptake was also lowest from cholate bile, although serosal secretion was highest. These results show that an increase in the biliary content of dihydroxy bile salts increases gall bladder permeability to both hydrophobic and hydrophilic molecules and may lead to the accumulation of lipids in the mucosa, as seen in cholesterolosis.