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Acute myocardial infarction (AMI) and heart failure (HF) are two major causes of cardiovascular mortality and morbidity. Early diagnosis of these conditions is essential to instigate immediate treatment that may result in improved outcomes. Traditional biomarkers of AMI include cardiac troponins and other proteins released from the injured myocardium but there are a number of limitations with these biomarkers especially with regard to specificity. In the past few years circulating nucleic acids, notably microRNA that are small non-coding RNAs that regulate various cellular processes, have been investigated as biomarkers of disease offering improved sensitivity and specificity in the diagnosis and prognostication of various conditions. In this review, the role of microRNAs as biomarkers used in the diagnosis of AMI and HF is discussed, their advantage over traditional biomarkers is outlined and the potential for their implementation in clinical practice is critically assessed.

Acute myocardial infarction (AMI) and heart failure (HF) are two major causes of cardiovascular mortality and morbidity. Early diagnosis of these conditions is essential to instigate immediate treatment that may result in improved outcomes. Traditional biomarkers of AMI include cardiac troponins and other proteins released from the injured myocardium but there are a number of limitations with these biomarkers especially with regard to specificity. In the past few years circulating nucleic acids, notably microRNA that are small non-coding RNAs that regulate various cellular processes, have been investigated as biomarkers of disease offering improved sensitivity and specificity in the diagnosis and prognostication of various conditions. In this review, the role of microRNAs as biomarkers used in the diagnosis of AMI and HF is discussed, their advantage over traditional biomarkers is outlined and the potential for their implementation in clinical practice is critically assessed.

Diabetic retinopathy (DR) is the leading cause of blindness in adults. Early detection and treatment of DR has been shown to reduce the risk of visual loss by as much as 90%. At present, there are no blood tests to detect DR. Previous studies have demonstrated the presence of nucleic acids in blood and raised levels of these markers have been reported in many conditions. The aim of this study was to evaluate the circulating levels of retina-specific mRNA in the assessment of DR.Blood samples were taken into PAXgene Blood RNA tubes from 89 diabetic patients and 19 healthy individuals. A reverse-transcription quantitative real-time PCR assay was used to measure circulating levels of mRNA for rhodopsin (Rho), retinal amine oxidase (RAO) and phosphodiesterase 6C (PDE6C). The results were normalized against mRNA for β-actin and total RNA. While mRNA for Rho, RAO and β-actin were detected in 100% of the subjects, PDE6C mRNA was only found in 60% of the individuals and melanopsin mRNA was not detected. When diabetic subjects were divided according to their DR status, significant differences were observed for Rho and RAO-Rho increased while RAO tended to decrease. The area under the curve ROC for Rho and Rho/RAO ratio to differentiate mild or no DR from significant DR (pre-proliferative and proliferative stages) were 0.756 and 0.823, respectively.These findings suggest that circulating mRNA may be useful in assessing DR.

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.

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.

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, P0.001). 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, P0.05). 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.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has been implicated in the pathogenesis of central obesity and metabolic syndrome. It has been shown that tissue-specific mRNA is present in blood, raising the possibility that quantifiable levels of 11beta-HSD1 mRNA may also be present in circulation. The aim of this study was to determine whether a relationship exists between circulating 11beta-HSD1 mRNA levels and various cardiovascular risk factors. Whole blood was collected using PAXgene Blood RNA tubes from 73 subjects. Total RNA was extracted, reverse-transcribed to cDNA, and 11beta-HSD1 mRNA was measured by quantitative real time PCR. The median BMI was 24 (range = 17-36) and the concentration of 11beta-HSD1 mRNA tended to decrease with increasing BMI (r=-0.206; P= 0.08). Multiple linear regression analysis identified BMI as an independent predictor of mRNA levels (beta=-0.412, P= 0.002). Other factors included in the analysis are age (beta= 0.952, P < 0.001), smoking (beta=-0.228, P= 0.027), alcohol intake (beta= 0.479, P < 0.001), and duration of menopause (beta=-1.602, P < 0.001). The results suggest a net decrease in systemic expression of 11beta-HSD1 with increased smoking, BMI, and duration of menopause. Increased systemic expression is seen with increasing age and alcohol intake.

The existence of circulating nucleic acids in plasma and serum (CNAPS) was first described almost six decades ago. However, the prognostic and diagnostic utility of this circulating DNA/RNA has only really begun to be appreciated in the last decade. Earlier studies concentrated mainly on investigations concerned with fetal medicine and oncology, and significant progress was made in both specialities. More recently the field of enquiry has extended further, and attention has turned to other pathologic states, including trauma, sepsis, myocardial infarction, stroke, transplantation, diabetes mellitus, and hematologic disorders. In some of these studies, mitochondrial as well as genomic DNA and tissue-specific mRNA have been analyzed, either quantitatively or qualitatively or both, and have been shown to be modified in the presence of disease. While there is tremendous potential for CNAPS as a clinical modality, many of the emerging studies seem to be confined to a few dedicated labs. Therefore, additional independent studies are necessary in some cases in order to show reproducibility, which will further consolidate the field. Despite this shortcoming, and with the evidently increasing number of applications for CNAPS, it is highly likely that routine testing, as described, will become reality within the next 5 to 10 years.

Diabetes mellitus is a major health problem across the world. Diabetic retinopathy (DR) and nephropathy are two of the major complications of diabetes. DR is the leading cause of blindness and diabetic nephropathy is the leading cause of end-stage renal failure. We have examined the potential value of circulating nucleic acids in the detection and monitoring of these two complications of diabetes. mRNA for nephrin was significantly higher in all diabetics compared to healthy controls and it was significantly higher in normoalbuminuric patients compared to healthy controls. This may indicate progression to microalbuminuric stage. Circulating rhodopsin mRNA was detectable in healthy subjects and in diabetic patients. It was significantly raised in diabetic patients with retinopathy. Higher rhodopsin mRNA in diabetic patients without retinopathy suggests that some of them may go on to develop it or already have it subclinically. Circulating nucleic acids have the potential to be noninvasive molecular tests for diabetic complications.

DNA and RNA fractions have been isolated from the whole blood, serum, plasma, the surface of blood cells, and urine of both healthy individuals and patients. The ability to isolate, quantify, and analyze these molecules has led to the identification of specific nucleic acid fragments related to particular disorders such as diabetes, cancer, myocardial infarction, and stroke, thereby permitting their early diagnosis. Currently, a number of methods for isolating the nucleic acids are employed and although a start has been made to compare the efficiencies of these methods, there is still a way to go before there are precise protocols for nucleic acid extraction. The older chemical methods of extraction still outperform some of the available kits. Some progress is being made to determine the origin of the circulating nucleic acids, although there are still many questions to be answered, including whether the source is through the spontaneous release of newly synthesized nucleic acid or whether it just derived from necrotic and apoptotic cells. In addition, it can be demonstrated that the nucleic acids can enter cells and exhibit a biological activity in the recipient cells. Hence, the question remains: Are the circulating nucleic acids freely entering tissues and cells from the blood and inducing changes in those tissues and cells? Further work is needed to elucidate these areas, and the various protocols must be standardized if the new methodology is to be widely and accurately applied in the diagnosis of disease and the monitoring of therapy. This chapter summarizes the work reported in this volume.

Follow-up of recurrent differentiated thyroid carcinoma involves the measurement of serum thyroglobulin (Tg). However, Tg autoantibodies are present in a high proportion of thyroid carcinoma patients (up to 25%) and these can interfere with the Tg immunoassays. To overcome this obstacle, investigators have used real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to measure Tg mRNA in the blood of patients with differentiated thyroid cancer, with varying degrees of success. In the present study, we demonstrate the first reported use of the PAXgene Blood RNA collection tube and extraction kit method for the preparation of RT-PCR-quality RNA with subsequent deployment of the latter in the development of a specific, sensitive, and reproducible Taqman assay for the detection and quantification of thyroglobulin mRNA. Beta-actin mRNA was also assayed and results are expressed as a ratio of Tg to beta-actin mRNA. The intra-assay coefficient of variations (CVs) for Tg and beta-actin mRNA assay were 27.7% and 25.4%, respectively. Inter-assay CVs were 20.8% and 28.8%, respectively, for the two assays. Tg mRNA was detected in all cancer subjects (n = 42) and healthy individuals (n = 20). Tg mRNA was significantly higher in cancer patients than in the healthy subjects (0.00169 +/- 0.00013 vs. 0.00051 +/- 0.00015; P

Diabetic retinopathy is the commonest complication of diabetes and is the biggest single cause of registered blindness in the UK. No biochemical tests exist to determine the precise state and rate of change of the eyes inthe diabetic patient. In the present study, using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we measured mRNA encoding the retina-specific pigment protein rhodopsin (RHO) in the peripheral blood of healthy individuals (n = 20) and diabetic patients (n = 46) with and without retinopathy. Beta-actin mRNA was also assayed and results are expressed as a ratio of RHO to β-actin mRNA. Peripheral blood was taken by venipucture directly into PAXgene Blood RNA collection tubes and RNA extracted by use of the PAXgene Blood RNA extraction kit, as per the manufacturer's (Qiagen) instructions. Diabetic patients were divided into three groups defined by the severity of retinopathy as assessed by fundoscopy: A, diabetic without retinopathy; B, background retinopathy; and C, preproliferative retinopathy. Medians of the ratios between groups were compared. RHO mRNA was successfully detected and quantified in peripheral blood in all healthy and diabetic groups, with levels shown to be significantly higher in diabetic patients than in healthy controls (2.54 X 10 - 5 vs. 1.29 X 10 - 5 ; P = 0.002). Significant differences in RHO mRNA levels were also seen between healthy control subjects and diabetic groups A (2.52 × 10 - 5 ; P = 0.022), B (1.98 X 10 - 5 ; P = 0.028), and C (5.08 × 10 - 5 ; P = 0.002). The results suggest that there is an increase in circulatory RHO mRNA with the severity of diabetic retinopathy.

Recent evidence suggests that cell-free plasma DNA has potential use as a prognostic marker in many clinical settings. The aim of the present study was to evaluate the prognostic role of cell-free plasma DNA in the prediction of clinical outcome in intensive treatment unit (ITU) patients. Cell-free plasma DNA was measured by real-time polymerase chain reaction assay for the beta-globin gene and SOFA score, APACHE II score, CRP concentrations, and clinical outcome (duration of stay, ventilation time, and mortality) were noted in 94 patients on admission to the ITU. The median plasma DNA concentration in ITU patients was 5493 GE/mL and this was significantly (P

Diabetes, which is a major health problem throughout the world is associated with increased mortality and morbidity. Early detection of those at risk and early detection of complications will help to reduce the adverse consequences. Current methods used for early detection of DM and its complications either have limitations or are expensive. Circulating miRNA and mRNA offer an exciting new approach to the diagnosis of diabetes and its complications. Several recent studies have reported that this approach is feasible. For example, mRNA for retinal specific proteins was reported to give good discrimination between different grades of diabetic retinopathy. In this chapter the use of miRNA and mRNA in diabetes and its complications is discussed.

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual a-isoforms of Na+, K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney α1, rabbit kidneyα1 and porcine cerebral cortex α3 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 0-100nmol/l) for ouabain with canineα1, and porcine α3 Na+, K+-ATPase showed similar inhibitory rofiles (IC50=15nmol/l), whilst rabbit α1 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 α-isoform and species selective. (Hypertens Res 2000; 23 Suppl: S45-S50)

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. β-actin mRNA was used for normalization. RPE65, retinoschisin, and β-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.

Previously it was shown that the circulating rhodopsin mRNA level was higher in diabetic retinopathy (DR). Recent evidence suggests that hypoxia may also be associated with DR. The aim of this study was to investigate the effect of oxygen desaturation on circulating retina-specific mRNA in type 2 diabetic patients. Thirty-five type 2 diabetic patients underwent overnight oximetry. Two parameters from oximetry were used to measure oxygen desaturation: the number of times per hour the oxygen saturation decreased by 4% or greater (number of dips/hr) and percentage of sleep time with oxygen saturation (SpO 2 )

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 β-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.

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.

Purpose Diabetic retinopathy (DR) is the leading cause of blindness among adults of working age in the UK. Early detection and appropriate management can prevent severe visual loss in 95% of cases. However, current screening methods are costly and suffer from subjective grading. Nucleic acids have been shown to circulate in plasma, with levels raised in conditions of cell death such as cancer and trauma. This study aimed to quantify circulatory retinal-specific nucleic acids, and to evaluate potential for their use in the assessment of DR. Methods Diabetic patients (n = 106) and healthy controls (n = 24) were recruited from the Diabetes and Ophthalmology departments at St Thomas’ Hospital. RNA was extracted from whole blood, with quantitative real-time RT-PCR used to quantify mRNA levels for rhodopsin and RPE65, two proteins expressed exclusively in the retina. Beta-actin mRNA was used for normalisation. Results Rhodopsin, RPE65 and beta-actin mRNA were detected in 100% of subjects. Circulating rhodopsin and RPE65 mRNA levels were higher in diabetic patients than healthy individuals (p < 0.02). Circulating rhodopsin mRNA was raised in all DR groups compared to healthy individuals, irrespective of presence or severity of DR (p < 0.02). With respect to healthy controls, circulating RPE65 mRNA levels were higher in diabetic patients with background and proliferative DR (p < 0.02). Patients with active proliferative DR (neovascularisation, vitreous and pre-retinal haemorrhage, or retinal detachment) possessed higher RPE65 mRNA and lower rhodopsin mRNA levels than those with quiescent disease (p < 0.01). Conclusion There is significant potential for use of these markers to screen for the presence of DR in diabetic patients in a quantitative manner using a blood test.

Kidney disease affects more than 20 million individuals in the United States alone. Although the causes of kidney failure are diverse, the glomerular filtration barrier is often the target of injury. Identification of the type of disease is important for targeting therapy. One area of particular interest is the ability to predict which patients with nephrotic syndrome might respond to steroids or cytotoxic therapies and which will not (1). Often this depends on renal biopsy, an invasive procedure with some risk of complications. A noninvasive test to determine the type of renal disease would be advantageous. During the past decade, nucleic acids have been detected in the circulation, and measurements of tissue-specific DNA and mRNA offer enormous potential for diagnosis and prognosis (2). Increased amounts of nucleic acids have been reported in disease, including lung cancer (3), thyroid cancer (4)(5), stroke(6), and trauma (7). We have recently shown that measurement of total DNA in plasma is a good prognostic marker of outcome in critically ill patients (8). In another study we observed that mRNA for rhodopsin is increased in diabetic retinopathy, and it may be a useful and inexpensive way to detect and monitor diabetic patients for retinopathy (9). Nephrin, a transmembrane protein with a large extracellular portion including 8 immunoglobulin-like domains, is expressed by visceral epithelial cells (podocytes) in the slit diaphragm of the glomerulus (10). A product of the NPHS1 gene located on chromosome 19, this protein is crucial for the integrity of the slit diaphragm, and abnormalities in this protein can lead to proteinuria and …

Diabetes mellitus is a major health problem throughout the world, and retinopathy is a serious and common complication of diabetes (1). Most individuals with diabetes will eventually develop diabetic retinopathy (DR). In the United States, up to 24 000 patients with diabetes become legally blind each year, and DR is the leading cause of blindness in adults 20–74 years of age (2). The United Kingdom Prospective Diabetes Study (UKPDS) showed that 39% of men and 35% of women had some degree of retinopathy at the time of diagnosis (3). With the incidence of diabetes mellitus increasing in many countries, the prevalence of DR as a major cause of blindness or visual impairment is also likely to increase. Factors influencing the development of retinopathy include type and duration of diabetes, degree of glycemic control, blood pressure, and serum lipids (3)(4)(5). Of these, duration of diabetes is the major factor related to the incidence of retinopathy. Clinical trials such as the Diabetic Retinopathy Study (DRS) (6) and the Early Treatment Diabetic Retinopathy Study (ETDRS) (7) have shown that effective laser photocoagulation could reduce the loss of vision by 90%. To be effective, regular screening for DR is required. Current recommendations are for annual screening for DR (8). Although recent studies suggest that in the absence of risk factors such as insulin use or duration of diabetes >20 years, screening every 2 or 3 years is adequate in type 2 diabetes (9). Despite these guidelines, 30–50% of patients with diabetes still do not get any eye examinations (10). Screening for DR includes visual acuity assessment as well as fundal photographs through dilated pupils. This involves cooperation between various healthcare professionals, such as diabetologists, optometrists, general practitioners, and photographic screening clinics. Patient eye status is classified according to a scale …

Recent evidence has shown elevated levels of cell-free plasma DNA in cancer patients. The aim of the present study was to quantify and compare the levels of cell-free plasma DNA in patients with prostate cancer, prostatic intraepithelial neoplasia (PIN), and benign prostatic hypertrophy (BPH) to examine if it offered a useful diagnostic test. Blood samples were obtained from 37 patients attending a clinic for prostate biopsies. Samples were taken prior to biopsy, within 1 hour of the biopsy, and then 2 weeks later. DNA was extracted using a QIAamp blood kit (Qiagen) and plasma DNA measured, in genome equivalents/milliliter plasma (GE/mL), using real-time quantitative PCR for the beta-globin gene. Prior to biopsy, plasma DNA concentration in BPH patients was 936 GE/mL (median; range: 633-2074 GE/mL), while cancer and PIN patients had significantly higher levels of DNA at 1734 GE/mL (median; range: 351-3131 GE/mL; P = 0.01) and 1780 GE/mL (median; range: 1514-2732 GE/mL; P = 0.04), respectively. Comparison of plasma DNA concentration before and after biopsy showed that 60 minutes after biopsy values were significantly higher in both BPH (1494 GE/mL; range: 613-2522 GE/mL; P = 0.029) and cancer (2758; range: 1498-5226 GE/mL; P = 0.007) patients. ROC analysis of the data indicated a sensitivity of 85% and a specificity of 73% when DNA concentration of 1000 GE/mL was taken as an indicator of malignancy or PIN. The data suggest that quantification of cell-free plasma DNA may have an important diagnostic role in distinguishing benign and malignant prostate disease.

Abstract The effect of carbidopa, a competitive inhibitor of dopamine synthesis on the excretion of sodium, dopamine (DA), and endogenous sodium transport inhibitor (ESTI), was examined in rats on normal and high salt diets for 7 days. ESTI activity was measured (1) as ouabain-like substance (OLS) by radioimmunoassay using an antibody raised against ouabain, (2) by inhibition of purified Na + ,K + -ATPase activity (ATPI), and (3) as digoxin-like substance (DLS) using a commercial digoxin assay. The OLS immunoassay was validated against rubidium transport studies. High salt diet caused an increase in OLS and ATPI but not DLS. Sodium excretion in rats on normal sodium intake was not affected by carbidopa treatment but was significantly lower in the high sodium diet group during the first 5 days of the study. In the low salt group, carbidopa treatment caused significant increases in OLS. We conclude that ESTI (measured as OLS) and DA are important in the natriuresis of salt loading.

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 (p0.001) 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.