Overcoming the effects of matrix interference in the measurement of urine protein analytes.

Using multiplex bead assays to measure urine proteins has a great potential for biomarker discovery, but substances in urine (the matrix) can interfere with assay measurements. By comparing the recovery of urine spiked with known quantities of several common analytes, this study demonstrated that the urine matrix variably interfered with the accurate measurement of low abundance proteins. Dilution of the urine permitted a more accurate measure of these proteins, equivalent to the standard dilution technique when the diluted analytes were above the limits of detection of the assay. Therefore, dilution can be used as an effective technique for over-coming urine matrix effects in urine immunoassays. These results may be applicable to other biological fluids in which matrix components interfere with assay performance.

Insulin's actions on plasma free fatty acids are normal in patients with stage 2 to 3 chronic kidney disease.

Resistance to insulin's action to suppress plasma nonesterified fatty acids (NEFA) is implicated in the hypertension and hyperlipidemia characterizing the metabolic syndrome. It is unknown whether insulin resistance to NEFA suppression is linked to hypertension and dyslipidemia in patients with mild chronic kidney disease (CKD). Eight patients with nonnephrotic, nondiabetic stage 2 to 3 CKD (I(125)-iothalamate clearances of 56 +/- 6 mL/min) and 7 hypertensive (HT) and 8 normotensive (NT) subjects with normal kidney function matched for age, gender, race, and percent body fat were studied. Plasma oleate, linoleate, palmitate, and stearate were measured during a 2-stage euglycemic hyperinsulinemic clamp procedure. Insulin suppressed plasma linoleate and oleate similarly in CKD (81%, 84%) and NT subjects (84%, 85%, respectively; P = NS) but less in HT patients (67%, 70%, P < .05 vs. CKD and NT). Likewise, the sum of NEFA were equally suppressed in the CKD and NT groups (P = NS) but not in HT subjects (P < .01 both vs. CKD and NT). Percent body fat correlated highly with NEFA suppression in the CKD and NT groups but not in HT subjects. Impairment of insulin's antilipolytic actions is not involved in the early pathogenesis of dyslipidemia and hypertension in patients with mild to moderated renal dysfunction.

Mechanism of lipid enhancement of alpha1-adrenoceptor pressor sensitivity in hypertension.

OBJECTIVE AND DESIGN: Plasma lipids enhance alpha1-adrenoceptor pressor sensitivity, impair baroreflex function, and correlate with increased blood pressure. This clinical study was designed to determine whether the enhanced alpha1-pressor sensitivity induced by acute hyperlipidemia is primarily mediated by increased vascular alpha1 responsiveness, reduced baroreflex sensitivity (BRS) or both. METHOD: Regional alpha1-adrenoceptor vasoreactivity was measured using a graded brachial artery infusion of the alpha1 agonist, phenylephrine, in seven subjects with stage 1 hypertension. Forearm blood flow was estimated from venous occlusion plethysmography. The phenylephrine dose-forearm blood flow response curve was used to determine alpha1-vascular reactivity (slope of the dose-response curve) and sensitivity, EC50 (phenylephrine dose inducing 50% maximal response). BRS (ms/mmHg) was measured as the slope of the progressive rise in systolic blood pressure and the resultant lengthening in the subsequent R-R interval after systemic intravenous boluses of phenylephrine. Subsequently, plasma lipids were raised with a 1-h systemic co-infusion of intralipid and heparin, after which measurements of regional vasoreactivity and BRS were repeated. RESULTS: Mean arterial pressure was 109 +/- 4 versus 110 +/- 3 (P = NS), vasoreactivity was -0.71 +/- 0.10 versus -0.82 +/- 0.10 (P = NS) and log EC50 was 1.47 +/- 0.29 versus 1.52 +/- 0.34 nmol/l (P = NS) before and after raising non-esterified fatty acids, respectively. In contrast, mean BRS was acutely reduced from 8.2 +/- 2.1 to 6.2 +/- 1.8 ms/mmHg (P = 0.02) after the lipid infusion. CONCLUSIONS: These findings suggest that in hypertensive patients, the primary mechanism for short-term alpha1-pressor hypersensitivity in response to hyperlipidemia is via the acute impairment of BRS.

Glomerular filtration rate can be accurately predicted using lean mass measured by dual-energy X-ray absorptiometry.

BACKGROUND: Accurate assessment of renal function is important in the management of patients with kidney disease yet is often difficult to obtain. Formulae, designed for clinical use, have been developed to predict glomerular filtration rate (GFR) utilizing serum creatinine (Scr). Additional parameters are included in these formulae to account for variations in Scr due to differences in total body lean mass in kg (LM). Therefore, the purpose of this study was to derive a simple formula to predict GFR based on Scr and direct quantification of LM. METHODS: Ten subjects with a wide range of renal function had GFRs determined by [125I]iothalamate clearance and LM determined by dual-energy X-ray absorptiometry as well as fasting measurements of Scr, serum and 24 h urine urea nitrogen, and albumin. RESULTS: The following formula was derived using LM (kg) and Scr (mg/dl): predicted GFR=(2.4xLM)-(0.75xLMxScr). The correlation coefficient for this formula was 0.97, when compared with [125I]iothalamate clearances, and similar to the MDRD formulae (R=0.87-0.95). CONCLUSION: Although further validation is necessary, these findings suggest that total body non-invasive measurement of LM along with Scr can be used to accurately predict GFR.

Prediction of urinary protein markers in lupus nephritis.

BACKGROUND: Lupus nephritis is divided into six classes and scored according to activity and chronicity indices based on histologic findings. Treatment differs based on the pathologic findings. Renal biopsy is currently the only way to accurately predict class and activity and chronicity indices. We propose to use patterns of abundance of urine proteins to identify class and disease indices. METHODS: Urine was collected from 20 consecutive patients immediately prior to biopsy for evaluation of lupus nephritis. The International Society of Nephrology/Renal Pathology Society (ISN/RPS) class of lupus nephritis, activity, and chronicity indices were determined by a renal pathologist. Proteins were separated by two-dimensional gel electrophoresis. Artificial neural networks were trained on normalized spot abundance values. RESULTS: Biopsy specimens were classified in the database according to ISN/RPS class, activity, and chronicity. Nine samples had characteristics of more than one class present. Receiver operating characteristic (ROC) curves of the trained networks demonstrated areas under the curve ranging from 0.85 to 0.95. The sensitivity and specificity for the ISN/RPS classes were class II 100%, 100%; III 86%, 100%; IV 100%, 92%; and V 92%, 50%. Activity and chronicity indices had r values of 0.77 and 0.87, respectively. A list of spots was obtained that provided diagnostic sensitivity to the analysis. CONCLUSION: We have identified a list of protein spots that can be used to develop a clinical assay to predict ISN/RPS class and chronicity for patients with lupus nephritis. An assay based on antibodies against these spots could eliminate the need for renal biopsy, allow frequent evaluation of disease status, and begin specific therapy for patients with lupus nephritis.