Andrew D. Baines

Production of urine free dopamine from DOPA; A micropuncture study

Abstract The contribution of free L-DOPA to urinary dopamine (D) was examined by microinjecting 3 H-DOPA into either proximal tubules or the peritubular space of innervated and denervated rat kidneys. Recirculation of radioactive material was corrected for by comparison with excretion of 14 C inulin included in the injectate. Urine radioactivity was characterized by HPLC. 69.4 ± 2.9% of 3 H-DOPA microinjected into proximal tubules appeard in urine from the ipsilateral kidney, 14.6 ± 1.6% of the 3 H was recovered as D. After microinjection into the subcapsular peritubular space 4 times as much 3 H appeared in the urine from the ipsilateral as from the contralateral kidney. Tubular secretion of total 3 H and 3 HD was calculated by comparison with 14 C inulin excretion. Chronically denervated kidneys secreted 36.0 ± 4.2% of the microinjected 3 H; innervated kidneys secreted 35.0 ± 3.0%. 3 H-D secretion was 14.5 ± 3.7% and 15.3 ± 2.8% of the total 3 H-DOPA microinjected into denervated and innervated kidneys respectively. We estimated that 30% of the urinary free D is derived from circulating free L-DOPA.

Digoxin reduces cardiac sympathetic activity in severe congestive heart failure

OBJECTIVES This study evaluated the effect of digoxin on cardiac sympathetic activity in patients with congestive heart failure. BACKGROUND Digoxin favorably alters autonomic tone in heart failure. Whether it reduces cardiac sympathetic drive in the setting of heart failure is unknown. METHODS Digoxin (0.25 mg intravenously) was administered to 12 patients with severe heart failure and elevated left ventricular end-diastolic pressure (> 14 mm Hg, Group A), 5 patients with less severe heart failure who had normal left ventricular end-diastolic pressure (> 14 mm Hg, Group B) and 6 patients with normal ventricular function. Seven additional patients with heart failure were studied as a time control group. Cardiac and total body norepinephrine spillover, systemic arterial pressure, left ventricular filling pressure and peak positive first derivative of left ventricular pressure were all assessed before and 30 min after administration of digoxin. RESULTS In Group A there were no changes in hemodynamic variables or total body norepinephrine spillover after digoxin administration; however, there was a significant reduction in cardiac norepinephrine spillover (263 +/- 70 to 218 +/- 62 pmol/min, mean +/- SEM, p < 0.001). In contrast, in Group B, digoxin caused a significant increase in cardiac norepinephrine spillover that was not associated with any hemodynamic changes or a change in total body spillover. There were no hemodynamic changes or a change in total body spillover. There were no hemodynamic or spillover changes in the time control or normal ventricular function group. CONCLUSIONS Digoxin, in the absence of detectable inotropic or hemodynamic effects, caused a reduction in cardiac norepinephrine spillover in patients with heart failure who had elevated filling pressures. This finding suggests a potentially beneficial primary autonomic action of digoxin in patients with severe heart failure.

Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells

Polyclonal antibodies were raised in rabbits against a 14-amino acid portion of the gibbon ape leukemia virus human membrane receptor Glvr-1. This epitope also contained seven amino acids common to the receptor for the amphotropic murine retrovirus Ram-1. Antibody specificity and molecular size of Glvr-1/Ram-1-related proteins were assayed by Western blot. Using a standard Laemmli buffer system, under reducing conditions, a single band of approximately 85 kDa (designated p85) was immunodetected in membranes prepared from opossum kidney (OK) cells and in brain membranes from rat, rabbit and hamster. In mouse brain, p85 as well as a protein of 70-72 kDa were immunodetected. This protein was also present in several other mouse tissues. Limited proteolysis of p85 and the 70-72kDa-protein from mouse yielded similar peptide fragments, suggesting that both proteins are related. Fragments of the same molecular masses were also detected in OK cell membranes following proteolysis, showing that p85 in both models (mouse brain and OK cell) share a similar sequence. p85 is not N-glycosylated since an assay using endoglycosidase F/N-glycosidase F did not alter the electrophoretic mobility of p85. We also observed that regulation of phosphate transport by incubating OK cells without any phosphate or by PTH treatment occurs without any changes in the amount of p85. In conclusion, these data demonstrate for the first time a Western blot detection of a type III phosphate transporter using polyclonal antibodies. They also suggest that, conversely to type I and type II phosphate transporters which are localized in the kidney, this third type of transporter is ubiquitous and probably absorbs the readily available phosphate from interstitial fluid for normal cellular functions in many species and tissues, serving as a housekeeping Na+/Pi cotransport system. This is also the first report showing that p85 is not regulated in the same manner as type II phosphate transporters.

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry. I. Preliminary considerations and analysis of rat liver and serum.

Interest in the biological behavior of a growing number of ele-ments, along with increasing recognition of the importance of interac-tions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improve-ments over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been real-ized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of tCP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, perfor-mance criteria, and certain problems peculiar to ICP-MS.

Nervous disorders of renal function.

Renal nerves contribute to the genesis of at least four disease processes. 1. Excess renal nerve activity contributes significantly to salt and water retention by patients with congestive circulatory failure. 2. Circumstantial evidence suggests that dopamine production may be deficient in a group of patients with idiopathic edema. Aldosterone secretion is high in this group and it has been shown that dopamine exerts a tonic inhibitory effect on angiotensin-stimulated aldosterone secretion. 3. Excess renal nerve activity probably plays a crucial role in the transition from hypotension and pre-renal failure to ischemic acute tubular necrosis. 4. Without doubt hyperactivity of renal nerves causes systemic hypertension in a variety of animal disease models. There is also good reason to believe that this occurs in some forms of human hypertension. The effects of the sympathetic nervous system on renal vascular resistance, renin release, tubular electrolyte reabsorption and aldosterone secretion are discussed in the context of these four diseases.

Variable selectivity of the Hitachi chemistry analyzer chloride ion-selective electrode toward interfering ions.

Chloride measurements by ion-selective electrodes are vulnerable to interference by anions such as iodide, thiocyanate, nitrate, and bromide. We have found that the degree of interference of these anions on the Hitachi chemistry analyzer chloride electrode varies from electrode to electrode and this variation can even occur within the same lot of membrane. This variation is not dependent upon the length of time the cartridge has been in the analyzer because no correlation existed between the usage time and the electrode response to interfering ions. Neither is this variation due to the deterioration of the electrode because all electrodes tested had calibration slopes within the manufacturers specification. Our study, however, showed that even after repeated exposure to a plasma sample containing 2 mM thiocyanate, the chloride electrode was still able to accurately measure the chloride in plasma without thiocyanate, thus confirming that a carryover effect does not exist from a previous thiocyanate-containing sample.

Use of inductively coupled plasma-mass spectrometry (icp-ms) for assessing trace element contamination in blood sampling devices

Abstract Inductively coupled plasma-mass spectrometry has been used to evaluate contamination of sampling and storage containers designated for use in the determination of minor and trace elements in body fluids. Potential contamination from Al, As, Au, B, Ba, Be, Bi, Cd, Co, Cr, Cs, Cu, Hg, Li, Mn, Mo, Ni, Pb, Pd, Pt, Rb, Sb, Sn, Sr, Ti, Tl, U, V, and Zn was evaluated. In general, plastic containers are preferable for sampling for most elements, and EDTA-containing collection tubes for whole blood are to be avoided. Systematic errors due to sampling of blood with stainless steel needles or Teflon catheters, into glass and plastic containers, have been estimated.

In patients receiving dopamine infusions for treatment of shock do free radicals convert dopamine to 6-hydroxydopamine?

Free hydroxyl radicals react with dopamine in vitro to produce the neurotoxin 6-hydroxydopamine (6-OHDA). If 6-OHDA were produced in vivo it could destroy central and/or peripheral neurons. Free radicals are produced in patients with cardiovascular or toxic shock. These patients are often treated with intravenous dopamine infusions. Using a newly developed method for measuring 6-OHDA in biological samples, we have examined blood from 15 patients receiving dopamine infusions for treatment of shock. 6-OHDA neither binds to nor elutes from either alumina or ion-exchange resins; therefore we used ODA-silica (Sep-Pak) to prepare samples for HPLC analysis with electrochemical detection. 6-OHDA disappeared rapidly from whole blood or serum at room temperature but was stable at -70 degrees C in an HClO4 extract. Recovery from Sep-Pak was 95% and detection limit 10 nmol/L. We repeated the experiments that showed in vitro hydroxyl radical conversion of dopamine to 5- and 6-OHDA. We detected neither 5- nor 6-OHDA in plasma samples from patients receiving dopamine infusions at 10-28 micrograms kg-1 min-1. Plasma dopamine in these patients was 1-5 mumol/L. We conclude that any 6-OHDA produced by hydroxyl radical attack in vivo is rapidly scavenged by endogenous substances containing sulfhydryl groups.