Ascorbate Improves Circulation in POTS

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doi: 10.1152/ajpheart.00018.2011301:H1033-H1042, 2011. First published 27 May 2011;Am J Physiol Heart Circ Physiol

Julian M. Stewart, Anthony J. Ocon and Marvin S. MedowsyndromeAscorbate improves circulation in postural tachycardia

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Ascorbate improves circulation in postural tachycardia syndrome

Julian M. Stewart,1,2 Anthony J. Ocon,2 and Marvin S. Medow1,2

Departments of 1Physiology and 2Pediatrics, New York Medical College, Valhalla, New York

Submitted 4 January 2011; accepted in final form 20 May 2011

Stewart JM, Ocon AJ, Medow MS. Ascorbate improves circula-tion in postural tachycardia syndrome. Am J Physiol Heart CircPhysiol 301: H1033H1042, 2011. First published May 27, 2011;doi:10.1152/ajpheart.00018.2011.Low flow postural tachycardiasyndrome (LFP) is associated with vasoconstriction, reduced cardiacoutput, increased plasma angiotensin II, reduced bioavailable nitricoxide (NO), and oxidative stress. We tested whether ascorbate wouldimprove cutaneous NO and reduce vasoconstriction when deliveredsystemically. We used local cutaneous heating to 42C and laserDoppler flowmetry to assess NO-dependent conductance (%CVCmax)to sodium ascorbate and the systemic hemodynamic response toascorbic acid in 11 LFP patients and in 8 control subjects (aged 23 2 yr). We perfused intradermal microdialysis catheters with sodiumascorbate (10 mM) or Ringer solution. Predrug heat response wasreduced in LFP, particularly the NO-dependent plateau phase (56 6vs. 88 7%CVCmax). Ascorbate increased baseline skin flow in LFPand control subjects and increased the LFP plateau response (82 6vs. 92 6 control). Systemic infusion experiments used Finometerand ModelFlow to estimate relative cardiac index (CI) and forearmand calf venous occlusion plethysmography to estimate blood flows,peripheral arterial and venous resistances, and capacitance before andafter infusing ascorbic acid. CI increased 40% after ascorbate as didperipheral flows. Peripheral resistances were increased (nearly doublecontrol) and decreased by nearly 50% after ascorbate. Calf capaci-tance and venous resistance were decreased compared with controlbut normalized with ascorbate. These data provide experimentalsupport for the concept that oxidative stress and reduced NO possiblycontribute to vasoconstriction and venoconstriction of LFP.

free radicals; vasoconstriction

POSTURAL TACHYCARDIA SYNDROME (POTS) accounts for mostcases of chronic orthostatic intolerance (21, 28, 39, 47, 48).POTS is defined by excessive increase in upright heart rate andsymptoms of orthostatic intolerance (39) improving with re-cumbence. Our laboratory (42) has described a subset of POTSdesignated low flow POTS (LFP) in which there is markedupright tachycardia and circulatory insufficiency even whilesupine including resting tachycardia, decreased cardiac output(CO), and decreased regional blood flows with peripheral andcutaneous vasoconstriction, findings often ascribed to a hy-peradrenergic state (1, 6). A consistent observation has beenincreased plasma angiotensin II (ANG II; Refs. 40, 43, 45),

which can act locally to decrease bioavailable nitric oxide(NO) by superoxide scavenging to create peroxynitrite (36).ANG II also acts through reactive oxygen species (ROS; Ref.35) to increase central and peripheral sympathetic activity (9,52). Both increased ANG II due to deficient ACE2 and de-creased bioavailable NO have been demonstrated in LFP dur-ing prior experiments using intradermal microdialysis tech-niques. Experiments made extensive use of the local vasodila-

tor heat response of nonglabrous skin with its three distinctphases: initial peak, nadir, and plateau. All phases, especiallythe plateau phase, are NO dependent (23, 30). The plateau canbe used as a bioassay for NO (44, 50) . All phases, especiallythe plateau phase are suppressed in LFP (29).

The importance of cutaneous oxidative stress became evi-dent from observations made in healthy volunteers in whomANG II-mediated attenuation of the local heat response wasrestored by intradermal perfusion of sodium ascorbate (46).Indeed, antioxidant treatment with ascorbic acid has beenshown to reduce excessive vasoconstriction related to oxidativestress in age and infirmity (19).

We hypothesized that when given cutaneously ascorbate

would improve the NO related local heating plateau in LFP,and when delivered systemically, ascorbate would reduce ex-cessive vasoconstriction in these patients.

METHODS

Subjects

To test these hypotheses, we studied POTS patients referred forevaluation of signs and symptoms of chronic orthostatic intolerancelasting 3 mo. Orthostatic intolerance was defined by symptomswhile upright relieved by recumbence. Symptoms included day-to-daydizziness, exercise intolerance, headache, fatigue, memory problems,nausea, blurred vision, pallor, and sweating. The diagnosis of POTSwas made during a tilt table test to 70 upright for a maximum of 10

min. POTS was diagnosed when there were symptoms of orthostaticintolerance during tilt associated with an increase in sinus heart rateexceeding 30 beats/min or to a rate exceeding 120 beats/min within 10min of tilt (28, 38). POTS patients were partitioned on the basis ofsupine calf blood flow into those who had LFP (1.2 ml100 mltissue1min1) and those that did not, using venous occlusion pleth-ysmography (42). For the current study, only LFP patients wereretained. The partitioning is not arbitrary but rather is based onoriginal data that showed a trimodal distribution of peripheral bloodflows in POTS (42). The LFP group appears to be physiologicallydistinct from the other subgroups with typically decreased absoluteblood volume (40), decreased cutaneous neuronal NOS activity (41),and increased plasma ANG II but not renin or aldosterone (40, 45).

Using these methods, we recruited 11 LFP patients (aged 19 24 yr,9 female, all Caucasian). Eight healthy volunteer subjects were also

recruited (aged 2026 yr, 6 female, all Caucasian) after a screeningupright tilt at 70 demonstrated normal orthostatic responses. Therewere no differences between the ages of the two groups. Volunteersubjects with a history of syncope or orthostatic intolerance wereexcluded.

Only subjects free from cutaneous, systemic, and cardiovasculardiseases were eligible. Subjects were not taking any medications andrefrained from alcohol and caffeine for 72 h before study. Therewere no smokers or trained athletes. Informed consent was obtainedand the Committee for the Protection of Human Subjects (InstitutionalReview Board) of New York Medical College approved all protocols.Female subjects were enrolled without regard to the phase of theirmenstrual cycle except that none were menstruating during testingprocedures.

Address for reprint requests and other correspondence: J. M. Stewart, Depts.of Pediatrics and Physiology, Center for Hypotension, Suite 1600, 19 Brad-hurst Ave., New York Medical College, Hawthorne, NY 10532 (e-mail:

[email protected]).

Am J Physiol Heart Circ Physiol 301: H1033H1042, 2011.First published May 27, 2011; doi:10.1152/ajpheart.00018.2011.

0363-6135/11 Copyright 2011 the American Physiological Societyhttp://www.ajpheart.org H1033


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Protocol 1

Cutaneous microdialysis of sodium ascorbate improves the local

heating response in LFP? Two microdialysis catheters were em-ployed. One was used to perfuse 10 mM sodium ascorbate dissolved

in lactated Ringer solution, and the other catheter contained Ringersolution and served as a control. Before microdialysis catheter inser-tion, laser Doppler flow (LDF) was measured over each insertion site

to estimate precatheterization laser Doppler blood flows. Laser probeswere removed and two microdialysis catheters were inserted and laserprobes positioned over the dialysis membranes. After recovery (seebelow), LDF was measured for 10 min while the catheters wereperfused with lactated Ringer solution to obtain baseline blood flows.Without moving the laser probes LDF was then recorded during local

heating of both catheters. A recovery period followed requiring3060 min until LDF reached baseline levels. We then perfused onecatheter with sodium ascorbate dissolved in Ringer and, after a 40-minloading period, verified LDF and repeated local heating while con-

tinuing ascorbate perfusion. The control catheter received Ringersolution only and underwent repeat local heating. After recovery fromthis second local heating, and as a final perfusion, 28 mM sodiumnitroprusside was delivered through each microdialysis catheter to

produce maximum blood flow.Instrumentation. Testing was at 25C2 h after a light breakfast.

Experiments were performed with the subject supine. The two cath-eters were placed 5 cm apart and inserted in the dermal space of theleft lateral calf after hair was gently removed from the insertion site.Each site was cooled with an ice-pack before catheter insertion toreduce discomfort. Each probe (MD-2000 Linear MicrodialysisProbes; Bioanalytical Systems, West Lafayette, IN) has a 10-mm

microdialysis membrane section that is placed in the intradermalspace using a 25-gauge needle as an introducer. The molecular masscutoff of the membrane is nominally 30,000 Da.

Following placement, both catheters were perfused with Ringer

solution at 2 l/min. An integrating LDF probe (Probe 413; Perimed,Stockholm, Sweden) containing seven individual probe tips wasplaced directly over each microdialysis catheter to measure LDF. LDF

was observed until flow recovered from the trauma of catheterinsertion. To estimate recovery, we used precatheterization laser

Doppler blood flows as a rough guide. Baseline LDF data werethereafter measured.

Once baseline LDF values were obtained and local heating re-sponse measured under untreated conditions, subjects received per-

fusate containing 10 mM sodium ascorbate dissolved in Ringer at arate of 2 l/min. The concentration of ascorbate was based on thework of Holowatz and Kenney (17).

Local heating. Once baseline LDF values were obtained, the areas

under each laser were heated at 1C/10 s to 42C for 30 min untila plateau was reached. Heat was then turned off to allow for recoveryto baseline LDF.

Monitoring. Heart rate was monitored by electrocardiography and

blood pressures were measured by finger plethysmography (Finom-

eter; TNO, Amsterdam, The Netherlands) of the right index or middlefinger intermittently recalibrated against oscillometry. Mean arterialpressure (MAP) was obtained by averaging the signal over 5 min. The

Finometer MAP was calibrated by oscillometry [using the formulaMAP (systolic pressure 2 * diastolic pressure)/3].

Protocol 2

Does systemic administration of ascorbic acid reduce peripheral

vasoconstriction and increase CO in LFP? INSTRUMENTATION AND

HEMODYNAMIC CALCULATIONS. On another day, tests began at 10AM after 4 h of fasting. Subjects refrained from caffeine for 72 h.Experiments were performed with the subjects supine. An intravenouscatheter was placed in the left antecubital vein.

1) Blood pressure, heart rate, and CO: we used the ModelFlowfeature of the Finometer to estimate relative change in CO. Relativetotal peripheral resistance (TPR) in Woods units (mmHgl1min) wascalculated as MAP/CO. Single lead electrocardiogram was obtainedfor heart rate and rhythm.

2) Impedance plethysmography: impedance plethysmography(IPG) can detect internal fluid volume shifts (32), during orthostaticstress (31). We used a four-channel digital impedance plethysmograph(THRIM; UFI, Morro Bay, CA) to measure volume shifts in fouranatomic segments designated the thoracic segment, the splanchnicsegment, the pelvic segment incorporating the lower pelvis to upperleg, and the leg segment. Ag/AgCl electrocardiographic electrodeswere attached to the left foot and hand, which served as currentinjectors. Additional electrodes were placed in pairs representinganatomic segments as follows: the ankle-upper calf just below theknee (leg), the knee-iliac crest (pelvic), the iliac crest-midline xyphoidprocess (splanchnic), and the xyphoid process to supraclavicular area(thoracic). We estimated the change in segmental fluid volume duringthe ascorbic acid infusion from the following formula:

Segmental blood volume (L R0)2R

where is the electrical conductivity of blood estimated as 53.2

exp(hematocrit 0.022) (10) and is assumed to be constant over thecourse of the experiment, R0 is the baseline resistance of a segment,and R is the change in resistance of that segment. Volume changesused for intergroup comparisons were calculated from maximumchanges in R during the infusion using the average baseline value of

R0 before ascorbate infusion and averaging over the entire change inresistance.

3) Forearm and calf hemodynamics: forearm and calf blood flow,venous pressure, and volume-pressure capacitance relationship weremeasured by venous occlusion plethysmography (VOP) using mer-cury in silastic, Whitney-type strain gauges (Hokanson, Bellevue,WA). Arm and thigh occlusion cuffs were placed around the upperand lower limbs 10 cm above the strain gauge. Forearm and calf bloodflows were obtained by rapidly inflating occlusion cuffs to a pressurejust below diastolic pressure to prevent venous egress. Inflating a

smaller secondary cuff to above systolic blood pressure briefly pre-vented wrist and ankle flow. Arterial inflow in units of ml/(100 mltissue)/min was estimated as the rate of change of limb cross-sectionalarea.

After recovery to baseline, we increased occlusion pressure grad-ually until a change in limb volume was detected. This representsresting venous pressure (Pv) (7). We calculated the forearm and calfperipheral arterial resistance in units of ml100 ml1min1mmHg1

from

mmHg (ml 100ml tissue) min from MAP-Pv)

flow

We also used VOP to estimate the pressure dependent forearm andcalf capacitance (volume-pressure) relation for comparison betweengroups, before and after ascorbic acid infusion. VOP measures vol-

ume changes in normalized units of ml/100 ml of tissue (7, 8).Resting venous volume (volume at Pv) was measured. With cuffs

deflated and the subject supine, we elevated the forearm and calf untilthere was no further decrease in limb size. Heart rate and bloodpressure remained unchanged suggesting that autonomic status wasunperturbed.

After a return to baseline, we used 10-mmHg pressure steps to amaximum of 60 mmHg to produce progressive limb enlargement.Pressure steps started at the first multiple of 10 exceeding Pv. Byfixing pressure with the cuff, the ascending volume-pressure relationwas obtained as shown in Fig. 1.

Separating filtration from vascular filling was performed. At lowerocclusion pressures, as shown in Fig. 1, the limb size reached aplateau. With higher pressures, an initial curvilinear change represent-

H1034 ASCORBATE AND POTS

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ing venous filling occurred, and thereafter limb volume increasedlinearly due to microvascular filtration. This is shown in Fig. 1, bottomright. With the use of least squares analysis (37), venous filling wasseparated from filtration by curve stripping the later linear portionleaving only the plateau-reaching curvilinear portion representingcapacitance vessel filling (left lower panel of Fig. 1). Occlusionpressure was maintained constant for 4 min to accomplish this.

The volume-pressure relation was computed. The volume-pressurerelation was constructed once the volume response was partitionedinto contributions from filling of capacitance vessels and fromfiltration.

The venous resistance was as computed. After stepwise increases inpressure were completed, the occlusion cuff was rapidly deflated. Theinstantaneous pressure difference between limb and central veins wasestimated by the final occlusion pressure (60 mmHg) ,and the central

Pv (assumed to be close to 0 mmHg). Venous resistance (Rv) wasestimated from the initial slope shown in Fig. 1, bottom right, usingthe formula Rv P(60 mmHg)/efflux (34).

IPG, VOP, blood pressure, EKG, and ModelFlow data were ac-quired continuously through an A/D conversion system at 200 Hz.

Protocol for Ascorbic Acid Infusion

Baseline data were collected for 10 min. Forearm and calf bloodflow was measured by VOP. Thereafter, we generated limb volume-pressure relationships. The cuffs were then rapidly deflated to calcu-late venous resistance.

After initial tests were completed and following a 30 min rest, allsubjects received 60 mg/(kg fat free mass) infusion of ascorbic aciddissolved in 100 ml of saline over 20 min followed by a maintenance

infusion of 20 mg/(kg fat-free mass) of ascorbic acid dissolved in 30ml saline administered over an hour. During the infusion bloodpressure, heart rate, ModelFlow, and IPG were measured continuouslywhile VOP blood flows were performed intermittently. After infusionswere complete, we repeated measurements of arterial pressure, Model-Flow, segmental blood volume changes by IPG, and VOP measure-ments of forearm and calf blood flow, Pv, capacitance measurements,and estimated venous resistance.

On another day, time and fluid volume control experiments wereperformed on three POTS patients and two control subjects. Baselinemeasurements were performed, and 130 ml of saline were infusedfollowing the same time and fluid volume schedule that was used forascorbic acid infusions. In both groups, hemodynamic parameterswere unchanged throughout the saline infusion.

Data and Statistical AnalysisCutaneous microdialysis perfusion of sodium ascorbate. LDF was

measured in perfusion units (pfu) to inform on cutaneous vascularconductance (CVC). CVC measurements were then converted to apercent maximum conductance (%CVCmax) by dividing CVC by themaximum CVC achieved after administration of 28 mM sodiumnitroprusside at the end of experiments.

Changes in baseline LDF before and after sodium ascorbate werecompared by paired t-test. Comparisons were made examining differ-ences between each phase of the local heating response pre- andpostascorbate infusion. Group comparisons of the heat response werealso averaged over POTS and controls subjects and graphicallydepicted. Data were obtained from individual heating curves beforeand after drugs.

Fig. 1. Representative supine leg plethysmographic tracing. Ascending portion of the volume-pressure relation was generated using 10-mmHg pressureincrements beginning at 20 mmHg as shown at top. Bottom left: curve stripping method used to separate intravascular filling from capillary filtration. Bottomright: decrease in limb blood volume that occurs immediately following the release of occlusion cuff pressure (at 60 mmHg). Initial flow is fit by a straight lineand used to compute venous resistance.

H1035ASCORBATE AND POTS



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Systemic administration of ascorbic acid. Unpaired t-tests withBonferroni correction were used to assess group differences between

control and POTS hemodynamics at baseline. Limb capacitance datawere compared between POTS and controls by two-way ANOVA forrepeated measures.

Pre- and postascorbate infusion data were compared by one-wayANOVA corrected for multiple comparisons. Since percent changesare shown, paired data were used. Capacitance curves were comparedbefore and after ascorbate between POTS and controls using multipleANOVA for repeated measures. Results are graphically depicted asmeans SE. The Statistical Package for the Social Sciences softwareversion 11.0 was used, and statistically significant differences arereported for P 0.05.

RESULTS

Cutaneous Microdialysis Perfusion of Sodium Ascorbate

Effects of sodium ascorbate on the local heating response.Table 1 and Fig. 2 show averaged %CVCmax measured alongthe heating curves. Data are reported for baseline, first thermalpeak, nadir, and plateau. Error bars have been omitted fromFig. 2 for clarity, but SE values are shown in the Table 1.Maximum conductance during Ringer perfusion was 2.16 0.13 pfu/mmHg for LFP compared with 2.25 0.13 pfu/mmHg for control where pfu refers to perfusion units of flowmeasured by LDF. There was no effect of ascorbate on themaximum CVC achieved with sodium nitroprusside: maxi-

mum conductance during Ringer perfusion was 2.31 0.16 forPOTS compared with 2.22 0.14 for control.BASELINE. Baseline %CVCmax in POTS was decreased com-pared with control subjects before ascorbate (P 0.05).Ascorbate similarly increased baseline %CVC in POTS andcontrol subjects.FIRST THERMAL PEAK. Before sodium ascorbate, the first thermal

peak %CVCmax was reduced in POTS compared with controlsubjects (P 0.01). The first thermal peak remained reducedin POTS compared with control subjects after ascorbate ad-ministration.NADIR. Before ascorbate, the nadir %CVCmax was similar inPOTS and control and increased (P 0.05) in POTS afterascorbate. POTS remained similar to control after ascorbate.PLATEAU. Before ascorbate, the plateau %CVCmax was reducedin POTS compared with control (P 0.001). After ascorbatethe plateau was unchanged in control subjects but increased inPOTS (P 0.001) to approximate the control subjects pla-teau. There was no change in the plateau of control subjectswith ascorbate.

Systemic Administration of Ascorbic Acid

Dimensions and preascorbic acid hemodynamic data. Preascor-bic acid data are shown in Table 2. POTS patients weighed lessthan control subjects (P 0.05) despite similar heights andhad reduced body mass indexes (P 0.025). The supine heartrate was increased in POTS compared with control (P 0.001). Arterial pulse pressure was reduced in POTS (P 0.05). Calf blood flow in LFP was decreased by definition. Calfarterial resistance was increased in POTS patients (P 0.001)while calf venous resistance was also increased in POTS (P 0.01). Both forearm and calf venous pressures were increasedin POTS compared with control (P 0.05).

Capacitance (volume-pressure) relation. The capacitance

relation before ascorbic acid infusion is shown in Fig. 3. Whileindividual forearm volumes at given pressures did not differindividually, there was a small overall decrease in calf capac-itance in POTS compared with control (P 0.05). There werehighly significant differences in calf capacitance (P 0.001)and reductions in volume at each pressure in POTS comparedwith control.

Effects of ascorbic acid on hemodynamic data. Hemody-namic data for a representative POTS patient during the infu-sion of ascorbic acid are shown in Fig. 4: CO and MAP

Table 1. Local heat response to sodium ascorbate(%CVCmax)

Control Subjects (n 8) P OTS Patie nts (n 11)

BaselineBefore ascorbate 9 1 6 1*After ascorbate 15 2 13 3

First thermal peakBefore ascorbate 62 8 42 6*After ascorbate 72 6 52 6*

NadirBefore ascorbate 37 5 30 4After ascorbate 44 4 43 3

PlateauBefore ascorbate 88 7 56 6*After scorbate 92 6 82 6

Values are means SE. %CVCmax, maximum percent cutaneous vascularconductance; POTS, postural tachycardia syndrome. *P 0.05, smaller thancontrol subjects. P 0.01, larger than preascorbate %CVCmax.

Fig. 2. Heating response expressed as percent maxi-mum conductance (%CVCmax) before and after ad-ministration of sodium ascorbate in control subjects(left) and in postural tachycardia syndrome (POTS)patients (right.). Data obtained before ascorbate isshown in black; data obtained after ascorbate perfu-sion is shown in gray. Ascorbate caused an increasein baseline conductance in POTS and control sub-

jects alike. There was no affect of ascorbate oncontrol plateau. Before ascorbate, POTS heating re-sponse was diminished compared with control butwas significantly increased after ascorbate.




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increased and TPR and heart rate decreased during the infu-sion. An increase in CO and decrease in TPR were observedin every POTS patient, but decreases in heart rate andincreases in MAP were not observed in every POTS patient.Individual POTS patients with findings similar to thoseshown in Fig. 4 exhibited a visible color change from pastywhite to pink, with warming of the peripheral extremities

and a sensation of overall warmth. Averaged percentchanges in hemodynamic data are shown in Table 3. Ascor-bate did not result in significant changes in hemodynamics incontrol subjects. However, in POTS patients pulse pressure in-creased (P 0.05), CI increased by 40% (P 0.01), while TPRdecreased (P 0.05). Forearm and calf blood flows increasedmarkedly (P 0.001) with a reciprocal decrease in arterialresistances (P 0.001). Forearm and calf venous resistances werealso reduced (P 0.01) in association with reduced venouspressures.

Regional blood volume changes in POTS are shown in Fig. 5.While small, they were significant with a shift of blood fromthe splanchnic (3 1%), pelvic (2 0%), and leg (2 1%) regional vasculature towards the thorax (7 1%), whichmay help to enhance CO.

Vascular capacitance was markedly affected by ascorbicacid infusion in POTS patients as depicted in Fig. 6, whichcompares pre- and postascorbate capacitance for all subjects.Ascorbate had minimal effect on control capacitances, butproduces a statistically significant overall increase of forearmcapacitance (P 0.05) and a larger increase of calf capaci-tance in POTS.

DISCUSSION

Key Findings

The current data show that at baseline LFP is associated

with decreased bioavailability of cutaneous NO, increasedcalf peripheral resistance, and reduced volume-pressure re-lationship under resting conditions in POTS compared withcontrol. Past work from our laboratory (42) using indicatordye techniques demonstrated reduced CO and increasedtotal peripheral resistance in similar LFP patients (42).These findings have been consistent across experiments anddifferent from results found in other POTS subgroups. Ourresults show improvement in CO and peripheral blood flowby the reduction of arterial and total peripheral resistance inLFP patients following ascorbic acid infusion. Afterloadreduction results in increased CO. Relatively small changesin regional blood volumes were observed with increased

Table 2. Dimensions and hemodynamics

Control Subjects (n 8) POTS Patients (n 11)

Age, yr 22 1.3 22.5 0.8Weight, kg 67 3 59.2 2*Height, cm 168 2 172 3Body mass index, kg/m2 24 0.9 20.1.5*Supine HR, beats/min 64 2 74 3*

Upright HR during HUT screen 86 4 124 5*Change in HR during HUT 23 2 49 3*Systolic BP, mmHg 120 2 113 4Diastolic BP, mmHg 62 2 66 3Pulse pressure, mmHg 57 2 48 3*Venous occlusion forearm blood flow, ml 100 ml1 min1 4.0 0.6 3.1 0.4Forearm arterial resistance, ml 100 ml1 min1 mmHg1 26 4 36 5Forearm venous pressure, Pv, mmHg 6 1 11 2*Forearm venous resistance, ml 100 ml1 min1 mmHg1 0.62.16 0.82.12Venous occlusion calf blood flow, ml 100 ml1 min1 3.0 0.4 1.0 0.2*Calf arterial resistance, ml 100 ml1 min1 mmHg1 32 4 66 8*Calf venous pressure, Pv, mmHg 11 1 16 2*Calf venous resistance, ml 100 ml1 min1 mmHg1 0.71.11 1.8 0.3*

Values are means SE. HR, heart rate; HUT, head-up tilt; BP, blood pressure. * P 0.05, smaller than control.

Fig. 3. Volume-pressure data points from supine POTS (gray) and control(black) subjects obtained from the forearm (top) and calf (bottom). Data wereobtained before ascorbate infusion. Data for the forearm were similar for POTSand control while there was a significantly reduced calf volume at givenpressure for POTS and thus decreased vascular capacitance. *P 0.05,compared with control.




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thoracic blood volume that could increase preload. Venousreturn and CO were clearly augmented by ascorbate andmay enhance peripheral blood delivery. Reduction of ve-nous resistance is also observed in LFP following ascorbateand facilitates peripheral venous return. Reduced venousresistance was associated with increased vascular capaci-

tance. While small compared with arterial resistance, ve-nous resistance can critically regulate pooling in the venousvasculature. Similar changes in capacitance and resistanceproperties were not observed in healthy volunteers receivingascorbic acid nor during time and volume control experi-ments, which ensured that ascorbate effects did not dependon exogenous volume loading. Cutaneous data showingincreased NO-dependent local heating response indicate thatpart of the overall effect of ascorbate possibly involves

improved bioavailability of NO in LFP. The results areconsistent with the hypothesis that ascorbate provides anantioxidant pronitrergic effect that improves circulatoryinsufficiency observed in LFP patients.

Dilator Properties of Ascorbate

Vasodilator and venodilator properties of ascorbic acid havebeen previously demonstrated. Thus, for example, high doseascorbic acid infusion abolishes the vasoconstriction of age inmen (19) and in estrogen-deficient postmenopausal women(33). Ascorbate can also modulate venous tone in humans (12).This is largely attributed to its antioxidant properties, whichcan exert its effects on an array of reactive oxidative speciesincluding superoxide and peroxides (3).

Fig. 4. Changes in mean arterial pressure(MAP; top left) and heart rate (HR; bottom left)and relative measures of cardiac output (CO;top right) and total peripheral resistance (TPR;bottom right) in a representative POTS patientduring the ascorbate infusion. Initiation ofascorbate infusion is indicated by the arrows.Woods U, Woods units; bpm, beats/min.

Table 3. Percent changes in hemodynamics with ascorbic acid

Percent Change Control Subjects POTS Patients

HR, beats/min 2 4 10 5Systolic BP, mmHg 4 6 11 5Diastolic BP, mmHg 0 5 1 5Pulse pressure, mmHg 1 4 10 3*Cardiac index, l min m2 4 3 40 14*Total peripheral resistance Woods units, mmHg l1 min 1 3 23 11*Venous occlusion forearm blood flow, ml 100 ml1 min1 2 3 30 5Forearm arterial resistance, ml 100 ml1 min1 mmHg1 2 1 25 10Forearm venous pressure, Pv, mmHg 0 3 21 7*Forearm venous resistance, ml 100 ml1 min1 mmHg1 10 8 37 9Venous occlusion calf blood flow, ml 100 ml1 min1 10 11 80 24*Calf arterial resistance, ml 100 ml1 min1 mmHg1 15 13 60 18*Calf venous pressure, Pv, mmHg 12 8 20 4*Calf venous resistance, ml 100 ml1 min1 mmHg1 10 13 44 12*

Values are means SE. *P 0.05, different from preascorbate.




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Vasoconstriction and Decreased NO Bioavailability in LFPThe vasoconstriction and potentially decreased NO bioavail-

ability of LFP can be explained by effects of ANG II excess.While ANG II can directly produce vasoconstriction (49),potent vasoconstrictive actions occur through ANG II type 1receptor (AT1R)-dependent interactions with NADPH oxidaseand other oxidases; these enhance production of ROS (11, 13,14), specifically superoxide, which scavenges NO to produceperoxynitrite. Thus, simultaneously, a potent oxidative/nitra-tive agent is produced while available NO is reduced (51).Increased ROS and specifically increased peroxynitrite oxidizetetrahydrobiopterin (BH4; Ref. 27), a vital cofactor for consti-tutive NOS. Absence of reduced BH4 uncouples NOS, causing

it to produce superoxide and diverse ROS (26). Once begun,the process results in positive feedback for oxidative stress andreduction in NO resulting in endothelial dysfunction and de-fective regulatory nitrergic pathways that adversely affectmyogenic tone in mesenteric artery and veins (2).

How Ascorbate Improves LFP

Ascorbic acid can interfere with this cycle of oxidativestress. At first it was thought to exert its primary effects onsuperoxide, but subsequent studies (3) have confirmed itsability to block lipid peroxidation. Ascorbate is a potent,perhaps the most potent, antioxidant against peroxynitrite-induced oxidation reactions (24), effectively quenching thenitrosation reaction (18) and restoring intracellular BH4 (19).

Mechanistic Predictions Based on the ROS Paradigm inLFP Patients

As proposed, the hypothetical pathophysiology of LFP de-pends on ANG II-induced superoxide formation, peroxynitritegeneration, and uncoupling of NOS through the removal of

BH4. The effects of ascorbic acid infusion and the cutaneouseffects of ascorbate support this hypothesis. Potential benefitmight derive from AT1R inhibition. To date, our pilot resultsusing single dose losartan have been disappointing althoughchronic testing is underway and may yield different outcomes.The hypothesis might also indicate benefit from BH4 supple-mentation, which is also under consideration. However, if theprimary defect is reduced ACE2, one wonders how that couldbe addressed. Decreased ACE2 expression occurs in heartfailure and is normalized by exercise. which also decreasesAT1R and increases NO synthesis (22). There is evidence thata vigorous program of exercise may improve POTS throughmechanisms that are not yet clear (4). While exercise training

Fig. 5. Percent changes in regional blood volumes produced by the infusion ofascorbic acid. There was a significant increase in thoracic blood volume inPOTS with reciprocal decreases in splanchnic, pelvic, and leg blood volumes.Significant changes were not observed in controls. *P 0.05, compared withzero.

Fig. 6. Volume-pressure relationship for fore-arms (top) and calfs (bottom) in POTS patients(right) and control subjects (left). Data ob-tained before ascorbate (pre-Asc) are shown

in black and after ascorbate (post-Asc) areshown in gray. On average, capacitance in-creased in forearms and markedly increased incalfs for POTS but not control subjects afterascorbate. *P 0.05, compared with control.




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clearly increases blood volume and while volume loadingoffers some palliative benefits in POTS, many of the pleitropiceffects of exercise remain to be determined.

Clinical Perspective

Patients with LFP present with a consistent phenotype of

circulatory insufficiency even while supine. This includes de-creased CO, increased peripheral vasoconstriction, cutaneouspallor, and tachycardia. Venous capacitance is reduced pre-sumably to accommodate this smaller blood volume. Ascor-bate improves these abnormalities in all patients. The pallid,cold skin grows warm and ruddy. Improvement of the venousvolume-pressure capacitance relation suggests that alterationsare not due to remodeling but rather to venoconstriction. Thusboth arterial vasoconstriction and peripheral venoconstrictionare enhanced in LFP and are remediated by ascorbate therapy.The literature suggests that vasoconstriction is mediated byreactive oxygen and reactive nitrogen species. Ascorbate ap-pears to rapidly reverse vascular constriction in acute patientsand may be proposed as a form of remedial therapy for LFP

patients. However, enterically administered ascorbic acid isexcreted in the urine nearly as rapidly as it is administeredpresenting a problem in obtaining plasma concentrations highenough to produce a vascular response. A blinded placebocontrol study of orally administered ascorbic acid that includesascorbate pharmacokinetics may therefore be clinically impor-tant. Further experiments comparing the effects of ascorbicacid across POTS subgroups are also indicated.

Limitations

There may be selection bias in comparing POTS patientswho have low calf blood flow measured by VOP, with controlsubjects who have a range of calf blood flows. Ideally, the most

informative study design would be to perform the currentexperiments on all POTS patients identifying baseline flow (orlow flow designation) as an a priori covariate and thencompare with a group of controls (with a similar covariate).

It has been suggested that we selected POTS patients simplyas those with the lowest flow values and that lower flow per sedictates the response to ascorbate. To address this possibility,we compared control subjects with lower than average calfblood flow to control subjects with greater than average calfblood flow. There were no significant differences in hemody-namic results between control subgroups while POTS resultswere different from controls. The smallest calf blood flow forcontrols was 1.7 ml100 ml1min1 compared with the largestcalf blood flow in LFP, which was 1.2 mlkg1min1. There

was no overlap. Also, although our original early studiesdescribed three subtypes using calf blood flow to distinguishamong physiological groups in POTS, the patients are nowwell characterized by an intermediate phenotype that includesreduced CO, reduced local heating plateau response, reducedblood volume, excessive resting arterial vasoconstriction, pal-lor, and often resting tachycardia and increased plasma ANG IIwhile supine compared with control subjects and to otherPOTS patients. While the term low flow POTS has becomethe convention used in our laboratory, it only describes oneaspect of this distinctive group of patients. Its continued use byus has allowed for a systematic evaluation of the complexfindings that we have described in this subset of POTS patients.

It also allows for comparisons between LFP and the otherphenotypes that we have described, namely normal and highflow POTS.

Our studies of hemodynamics were technically limited.While VOP afforded us the ability to accurately estimatechanges in venous and arterial properties in the regional fore-arm and calf circulations, and while impedance methods of-

fered additional information concerning thoracic, pelvic, andsplanchnic blood volume shifts, equivalent volume-pressurerelations could not be obtained. Also, impedance measure-ments lack anatomic specificity regarding liver, intestinal,renal, and other hemodynamically important circulations. Gen-eralizing from the specific to the general is risky. However,changes in CO and TPR were consistent with changes in armand leg blood flows, suggesting that similar phenomena mayoccur system wide.

We did not measure plasma ascorbate levels; however, wefollowed the same intravenous ascorbic acid protocol em-ployed successfully by Jablonski et al. (20).

We did not directly measure NO, ROS, or reactive nitrogenspecies. We do have past experience with attempted directmeasurements of NO in skin and in blood using the chemilu-minescence method. Skin NO was highly variable and inter-pretable only by retaining nitrite and discarding nitrate. BloodNO was even more variable because of hemoglobin scavengingof NO. Cutaneous microdialysis is a verified surrogate forsystemic responses, and the plateau phase of the local heatresponse is a well-defined bioassay for NO (23, 30). Theobjective of performing cutaneous microdialysis was to dem-onstrate that ascorbate increases the NO-dependent heatingplateau and that therefore NO bioavailability was increased byascorbate in LFP. We also did not measure ROS. This isdifficult to accomplish in humans although attempts are under-way. Our past data indicate ANG II excess (40, 43) and

decreased NO bioavailability (41) in LFP, consistent withliterature (11, 14) in which ANG II induced superoxide over-production scavenges NO producing peroxynitrite that in turndecreases BH4 further reducing NO formation via NOS un-coupling(15).

Investigators as well as subjects were not blinded althoughlimited blinding of subjects occurred during saline placeboadministration. Absent blinding weakens our conclusions.

We studied mostly females without regard to menstrualcycle. The phase of the menstrual cycle can exert importanteffects on NO-dependent mechanisms. There is also evidenceindicating differences in renin activity and serum aldoste-rone but not in CO, stroke volume, blood pressure, heartrate, or total peripheral resistance in the midluteal phase

compared with the early follicular phase of the menstrualcycle in POTS (5).

LDF measures relative changes in skin blood flow andconductance. For example, if the maximum absolute conduc-tance during sodium nitroprusside dialysis is similar for dif-ferent subjects, then relative changes are thought to reflect truevascular properties. However, changes in the numbers of mi-crovessels or their properties, differences in skin color, andambient temperature fluctuations can all exert effects on themeasured maximum flow such that two patients with similarCVCmax may have different vascular properties.

Cooling the skin to help subject comfort before insertion ofthe catheter may affect blood flow and CVC measurements.




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However, there is no way to penetrate the skin without causinga response of some sort. Thus, for example, the act of cathe-terization changes blood flow response characteristics. Anyand all attempts to reduce the pain of catheterization that isnecessary for ethical reasons also changes the blood flow. Thisissue has been addressed in a study by Hodges et al. (16) inwhich the investigators found that maximum conductance in

response to whole body heating was not affected by ice or localanesthesia but was decreased if no form of pain reduction wasused.

Finally, the model proposed while consistent with the datahas therapeutic limitations. Large doses of intravenous ascor-bic acid were administered and can easily exceed the bloodconcentration range (not measured in these experiments)achievable by oral administration of ascorbic acid. Ascorbate istypically excreted in the urine more rapidly than it can beabsorbed from large enteric doses.

ACKNOWLEDGMENTS

We thank members of the New York Medical College Department ofPediatrics, especially its Chairman, Dr. Leonard Newman, and the Division of

Pediatric Cardiology, especially its Director, Dr. Michael H. Gewitz, forunflagging support. We also acknowledge our intellectual debt to our mentorsDr. Thomas H. Hintze, Dr. Gabor Kaley, Dr. David Robertson, and Dr. PhillipLow for constant inspiration and stimulation.

GRANTS

This work was supported by the National Heart, Lung, and Blood InstituteGrants 1-RO1-HL-074873, 1-RO1-HL-087803, and 1-F30-HL-097380.

DISCLOSURES

We have nothing to disclose concerning any potential conflict of interest(e.g., consultancies, stock ownership, equity interests, patent-licensing ar-rangements, lack of access to data, or lack of control of the decision topublish).

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Ascorbate Improves Circulation in POTS