Integrated Microfluidic Aptasensor for Mass Spectrometric Detection of Vasopressin in Human Plasma Ultrafiltrate.

We present a microfluidic aptamer-based biosensor for detection of low-molecular-weight biomarkers in patient samples. Using a microfluidic device that integrates aptamer-based specific analyte extraction, isocratic elution, and detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we demonstrate rapid, sensitive and label-free detection of arginine vasopressin (AVP) in human plasma ultrafiltrate. AVP molecules in complex matrices are specifically captured by an aptamer that is immobilized on microbeads via affinity binding in a microchamber. After the removal of unbound, contaminating molecules through washing, aptamer-AVP complexes are thermally disrupted via on-chip temperature control. Released AVP molecules are eluted with purified water and transferred to a separate microchamber, and deposited onto a single spot on a MALDI plate via repeated, piezoelectrically actuated ejection, which enriches AVP molecules over the spot area. This integrated on-chip sample processing enables the quantitative detection of low-abundance AVP by MALDI-TOF mass spectrometry in a rapid and label-free manner. Our experimental results show the detection of AVP in human plasma ultrafiltrate as low as physiologically relevant picomolar concentrations via aptamer-based selective preconcentration, demonstrating the potential of our approach as a rapid (~ 1hr), sensitive clinical AVP assay.

Vasopressin administration facilitates fluid removal during hemodialysis.

Inadequate secretion of vasopressin during fluid removal by hemodialysis may contribute to the cardiovascular instability that complicates this therapy and administration of exogenous hormone, by supporting arterial pressure, may facilitate volume removal. To test this, we measured plasma vasopressin in patients with end-stage renal disease (ESRD) during hemodialysis and found that despite significant fluid removal, plasma vasopressin concentration did not increase. We further found that ESRD did not alter the endogenous removal rate of plasma vasopressin and that plasma hormone is not dialyzed. Finally, in a randomized, double-blinded, placebo-controlled trial in 22 hypertensive patients, we examined the effect of a constant infusion of a non-pressor dose of vasopressin on the arterial pressure response during a hemodialysis in which the target fluid loss was increased by 0.5 kg over the baseline prescription. We found that arterial pressure was more stable in the patients receiving vasopressin and that while only one patient (9%) in the vasopressin group had a symptomatic hypotensive episode, 64% of the patients receiving placebo had such an episode (P=0.024). Moreover, increased fluid removal was achieved only in the vasopressin group (520+/-90 ml vs 64+/-130 ml, P=0.01). Thus, administration of non-pressor doses of vasopressin to hypertensive subjects improves cardiovascular stability during hemodialysis and allows increased removal of excess extracellular fluid. Inadequate vasopressin secretion during hemodialysis-induced fluid removal is a likely contributor to the intradialytic hypotension that limits fluid removal.

Blood pressure instability during hemodialysis.

Most patients with end-stage renal disease (ESRD) maintained on hemodialysis have chronic hypertension. However, hypotension is a frequent complication of hemodialysis, probably because of impaired baroreflex function. Less frequently, increases in pressure can be a complication of hemodialysis. Detailed studies of patients with these abnormalities in arterial pressure during hemodialysis may yield insights into the regulation of arterial pressure during ESRD.

Voluntary reciprocal altruism: a novel strategy to encourage deceased organ donation.

New strategies are needed to encourage organ donation. Altruism, the impulse that underlies our present system, is undermined by proposals that provide tangible inducements to improve donation which are, in their own subtle ways, coercive. I propose a new strategy based on implementing an option to donate that reinforces the strong reciprocity which drives anonymous altruism.

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

Aptamer-based folding fluorescent sensor for cocaine.

We adapted in two steps a deoxyribonucleotide-based aptamer to signal the recognition of cocaine: an instability was engineered in one stem of a three-way junction that forms the cocaine-binding pocket and the resulting short stem was end labeled with a fluorophore and a quencher. In the absence of cocaine, two stems are open, but in its presence they close and the three-way junction forms. This major structural change brings fluorophore and quencher together thereby signaling the presence and concentration of ligand. The sensor is selective for cocaine over its metabolites, can operate in serum, and is useful for the screening of cocaine hydrolases.

A catalytic antibody against cocaine attenuates cocaine's cardiovascular effects in mice: a dose and time course analysis.

The murine monoclonal antibody 15A10 (mAb 15A10), elicited by a transition-state analog for cocaine hydrolysis, has previously been shown to metabolize cocaine in vitro and in vivo. The present experiments were designed to evaluate further the in vivo effectiveness of mAb 15A10 in blocking cardiovascular effects of acute cocaine administration. Balb/c mice were implanted with a femoral artery catheter utilized for mean arterial pressure (MAP) monitoring, and administered intravenous (i.v.) pretreatments of either mAb 15A10 (10, 32, 100 and 300 mg/kg) or vehicle prior to cocaine injection (100 mg/kg, i.p.). A time course analysis for mAb 15A10's effect was also conducted, for which either vehicle or 100 mg/kg mAb 15A10 was infused 1, 3, 10 and 30 days prior to cocaine treatment. During the cardiovascular recording sessions, mice were awake and freely moving within a limited area. Increases in MAP (approximately 25 mm Hg) following cocaine injection were dose-dependently attenuated by mAb 15A10. The antibody-attenuated cocaine-induced increases in MAP at 1- and 3-day pretreatment times, and reduced mortality at some of the time points studied. With 100 mg/kg antibody, plasma cocaine levels were significantly decreased early in the recording session, whereas levels of ecgonine methyl ester increased significantly. Although 10-fold greater quantities of antibody are required to observe significant effects in mouse, compared to our previous studies in rats, the present mouse model provides a convenient paradigm for investigating catalytic and non-catalytic antibodies.

Anticocaine catalytic antibodies have no affinity for RTI compounds: implications for treatment.

Potential medications for cocaine abusers include: anticocaine catalytic antibodies, which could serve as circulating peripheral blockers of cocaine that prevents its action in the brain; and 3-phenyltropane cocaine analogs, which could serve as potent, selective, and long-lasting substitutes that reduce drug-seeking. In order to evaluate the compatibility of these agents, we measured if a catalytic antibody would bind and interact with some cocaine analogs. Anticocaine catalytic antibody 15A10 had no significant affinity for RTI-51, RTI-112, or RTI-177 as examined by ELISA. They exhibited high affinity for the immunogen TSA1 in the same experiment, as expected. Because the antibody and the RTI compounds do not interact, they are candidates for simultaneous use.

Titer-dependent antagonism of cocaine following active immunization in rhesus monkeys.

Immunization may be a useful pharmacokinetic antagonist therapy for cocaine users. Three rhesus monkeys were immunized with a cocaine:bovine serum albumin conjugate in alum and later with complete and incomplete Freund's adjuvants. Monkeys developed cocaine-binding antibodies (as measured by enzyme-linked immunosorbent assay) after immunization with alum; greater antibody titers developed after immunization with Freund's adjuvants. The response rate-decreasing effect of cocaine diminished in proportion to antibody titer; there was no substantial change in the rate-decreasing effect of bupropion. Plasma cocaine concentrations increased in proportion to antibody titer. Immunizations were well tolerated and had no effect on response rates. These data suggest that the antibody response to a cocaine antigen can produce a specific pharmacokinetic shift in cocaine distribution sufficient to antagonize a behavioral effect of the drug, and can do so with minimal side effects.

Catalytic molecular beacons.

We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.

Natural and artificial enzymes against cocaine. I. Monoclonal antibody 15A10 and the reinforcing effects of cocaine in rats.

Recent reports have indicated the potential usefulness of anticocaine catalytic monoclonal antibodies in reducing cocaine's toxic and reinforcing effects by altering its pharmacokinetics to favor increased metabolism to the systemically inert products ecgonine methylester and benzoic acid. The present study was designed to further these findings by evaluating the hypothesis that administration of the anticocaine catalytic monoclonal antibody mAb 15A10 would dose and time dependently reduce behavior maintained by a range of doses of i.v. cocaine. Male Sprague-Dawley rats were trained in daily 8-h sessions to self-administer i.v. cocaine. A within-session multiple-dose protocol was used wherein rats were allowed access to saline or one of six doses of cocaine [0 (saline), 0.015, 0.03, 0.06, 0 (saline), 0.125, 0.25, or 0.5 mg/kg/injection] each hour in the order stated. After demonstrating stable dose-response curves over 3 consecutive days, rats were given 30-min pretreatments of saline or mAb 15A10, (10, 30, or 100 mg/kg i.v.). Antibody, but not saline, pretreatments significantly altered dose-response curves for cocaine self-administration in a dose- and time-dependent manner, resulting in downward and rightward shifts in rates of responding across the cocaine dose range. These effects were apparently not attributable to general behavioral suppression, because operant behavior for an alternative reinforcer was not likewise affected. The present data extend previous work indicating that pharmacokinetic approaches may be of worth in the search for clinically effective cocaine antagonists.

Homogeneous assays based on deoxyribozyme catalysis.

We report herein the first homogeneous assays based on the ribonuclease activity of a deoxyribozyme. The previously reported deoxyribozyme was covalently modified with biotin and used to assay biotin-binding interactions through changes in fluorescence upon substrate turnover. Deoxyribozymes with fluorescence-based reporting have the potential to serve as general analytical tools.

Application of artificial enzymes to the problem of cocaine.

Cocaine mediates its reinforcing and toxic actions through a "loss of function" effect at multiple receptors. The difficulties inherent in blocking a pleiotropic blocker pose a great obstacle for the classical receptor-antagonist approach and have contributed to the failure-to-date to devise specific treatments for cocaine overdose and addiction. As an alternative, we have embarked on an investigation of catalytic antibodies, a programmable class of artificial enzyme, as "peripheral blockers"--agents designed to bind and degrade cocaine in the circulation before it partitions into the central nervous system to exert reinforcing or toxic effects. We synthesized transition-state analogs of cocaine's hydrolysis at its benzoyl ester, immunized mice, prepared hybridomas, and developed the first anti-cocaine catalytic antibodies with the capacity to degrade cocaine to non-reinforcing, non-toxic products. We subsequently identified several families of anti-cocaine catalytic antibodies and found that out most potent antibody, Mab15A10, possessed sufficient activity to block cocaine-induced reinforcement and sudden death in rodent models of addiction and overdose, respectively. With the potential to promote cessation of use, prolong abstinence, and provide a treatment for acute overdose, the artificial enzyme approach comprehensively responds to the problem of cocaine.

Vasopressin in the treatment of milrinone-induced hypotension in severe heart failure.

The use of phosphodiesterase inhibitors such as milrinone in the treatment of severe heart failure is frequently restricted because they cause vasodilation and hypotension. In patients with decompensated heart failure with hypotension after treatment with milrinone, low doses of vasopressin restored blood pressure without inhibiting the inotropic effect of milrinone.

Vasopressin as an alternative to norepinephrine in the treatment of milrinone-induced hypotension.

OBJECTIVE: To determine whether vasopressin could be effective in treating the hypotension associated with phosphodiesterase III inhibition. Phosphodiesterase III inhibitors are cardiotonic agents that increase myocardial contractility and decrease vascular smooth muscle tone. The vasodilatory effect can be profound, and the resulting hypotension frequently requires the administration of catecholamine pressors. DESIGN: Retrospective analysis of existing data. SETTING: The medical or surgical intensive care unit of Columbia-Presbyterian Medical Center. PATIENTS: Three consecutive patients receiving milrinone and requiring catecholamine pressors to maintain systolic arterial pressure of > or =90 mm Hg. INTERVENTIONS: Vasopressin was administered to the three patients. MEASUREMENTS AND MAIN RESULTS: Vasopressin (0.03-0.07 units/min) increased systolic arterial pressure from 90+/-4.7 to 130+/-2.3 mm Hg while reducing the administration of catecholamine pressors. CONCLUSIONS: Vasopressin at very low doses appears to be an effective vasopressor for milrinone-induced hypotension.

Arginine vasopressin in the treatment of 50 patients with postcardiotomy vasodilatory shock.

BACKGROUND: The barroreflex-mediated secretion of arginine vasopressin has been found to be defective in a variety of vasodilatory shock states, such as postcardiotomy shock, and administration of the hormone markedly improves vasomotor tone and blood pressure. The high incidence of vasodilatory shock in patients undergoing left ventricular assist device (LVAD) implantation makes this population an ideal model in which to assess the risks and benefits of vasopressin. METHODS: The medical records of the 102 patients receiving LVADs at Columbia-Presbyterian Medical Center from January 1995 to August 1998 were reviewed. Fifty patients were eligible for study based on a history of arginine vasopressin administration in the operating room or intensive care unit within 24 hours of implantation. RESULTS: Despite LVAD implantation and the administration of vasopressors, patients were hypotensive with a mean arterial pressure less than 60 mm Hg. The administration of vasopressin (0.09+/-0.05 U/min) increased mean arterial pressure (58+/-13 to 75+/-14 mm Hg; p<0.001) while reducing norepinephrine administration (11.7+/-13 to 7.9+/-6.0 mcg/min; p = 0.023). There was no significant change in LVAD flow. The incidence of compromised regional perfusion was not different between LVAD patients who received vasopressin as compared to hemodynamically stable LVAD patients who did not receive vasopressin. CONCLUSIONS: We have demonstrated vasopressin at low doses to be a safe and an effective vasopressor in 50 patients with postcardiotomy vasodilatory shock.

Intravenous arginine-vasopressin in children with vasodilatory shock after cardiac surgery.

BACKGROUND: Recent investigations at our institution have studied a variety of vasodilatory shock states that are characterized by vasopressin deficiency and pressor hypersensitivity to the exogenous hormone. Our experience in adults prompted the use of arginine-vasopressin (AVP) in a similar group of critically ill children. METHODS AND RESULTS: This report describes our early experience (from February 1997 through April 1998) in 11 profoundly ill infants and children (5 male, 6 female) ages 3 days to 15 years (median, 35 days) treated with AVP for hypotension after cardiac surgery which was refractory to standard cardiopressors. Although underlying heart disease was present (congenital heart defects in 10 and dilated cardiomyopathy in 1), only 2 patients had severely depressed cardiac function as demonstrated by 2D echocardiogram before administration of AVP. All patients were intubated and receiving multiple catecholamine pressors and inotropes, including dobutamine (n=10), epinephrine (n=8), milrinone (n=7), and dopamine (n=4) before receiving AVP. Five patients received AVP intraoperatively immediately after cardiopulmonary bypass, 5 in the intensive care unit within 12 hours of surgery, and 1 on postoperative day 2 for hypotension associated with sepsis. The dose of AVP was adjusted for patient size and ranged from 0.0003 to 0.002 U. kg(-1). min(-1). During the first hour of treatment with AVP, systolic blood pressure rose from 65+/-14 to 87+/-17 mm Hg (P<0. 0001; n=11), and epinephrine administration was decreased in 5 of 8 patients and increased in 1. Plasma AVP levels before treatment were available in 3 patients and demonstrated AVP depletion (median, 4.4 pg/mL; n=3). All 9 children with vasodilatory shock survived their intensive care unit stay. The 2 patients who received AVP in the setting of poor cardiac function died, despite transient improvement in blood pressure. CONCLUSIONS: Infants and children with low blood pressure and adequate cardiac function after cardiac surgery respond to the pressor action of exogenous AVP. AVP deficiency may contribute to this hypotensive condition.

Vasopressin deficiency and pressor hypersensitivity in hemodynamically unstable organ donors.

BACKGROUND: Solid organ donors often develop hypotension due to vasodilation, and recently we observed that a variety of vasodilatory states are characterized by vasopressin deficiency and hypersensitivity. Thus, we investigated the prevalence of vasopressin deficiency in hypotensive solid organ donors without clinical evidence of diabetes insipidus; we also investigated the vasopressor effect of vasopressin replacement in hypotensive donors. METHODS AND RESULTS: Fifty organ donors were evaluated for hemodynamic instability, (mean arterial pressure [MAP]

Low-dose vasopressin in the treatment of vasodilatory septic shock.

BACKGROUND: Despite appropriate therapy, refractory hypotension often occurs in septic shock. A double-blinded placebo controlled clinical trial was performed to assess the role of low-dose vasopressin (VP) as a pressor agent in septic shock. METHODS: Patients admitted to a trauma intensive care unit with vasodilatory septic shock were randomized to receive either VP at 0.04 U/min (n = 5) or placebo (n = 5). Vasodilatory septic shock was defined as a need for catecholamine agents to maintain a mean arterial pressure more than or equal to 70 mm Hg, despite a cardiac index more than 2.5 L/min and a minimal pulmonary artery wedge pressure more than 12 mm Hg. After 1 hour of initiation of the study drug, attempts to discontinue norepinephrine, phenylephrine, and/or dopamine, in respective order, were undertaken provided that the mean arterial pressure remained more than or equal to 70 mm Hg. RESULTS: A vasopressin infusion increased systolic arterial pressure (98 +/- 5 to 125 +/- 8 mm Hg, p < 0.008) because of peripheral vasoconstriction (systemic vascular resistance increased from 878 +/- 218 to 1,190 +/- 213 dynes/s per cm(-5) p < 0.05). Arterial pressure and systemic vascular resistance were statistically unaffected in the placebo group. Before study termination, measured at 24 hours after drug initiation, two patients in the placebo group died of refractory hypotension. However, all patients receiving VP survived the 24-hour study period and had all other catecholamine pressors withdrawn and blood pressure maintained solely with a low-dose VP infusion. CONCLUSION: A VP infusion improved arterial pressure and permitted the withdrawal of catecholamine vasopressors. VP is a useful agent in the treatment of refractory septic shock.