We studied 12 patients with autonomic insufficiency and orthostatic hypotension (OH), all with familial amyloid polyneuropathy (FAP). FAP was diagnosed on the basis of clinical findings, a family history of FAP, the presence of the TTR Met30 mutation in plasma, and a positive skin biopsy for amyloid. These patients with autonomic insufficiency were classified as the OH group. Twelve patients without orthostatic hypotension, but with frequent, typically repetitive neurally mediated (NM) syncope episodes and with a positive head-up tilt test that induced symptoms, were classified as the NM group. The same protocol was also performed in 10 patients with florid postural tachycardia syndrome (POTS), with orthostatic tachycardia at more than 120 bpm2; these were classified as the POTS group.

Twelve apparently healthy normotensive volunteers were used as controls and classified as the control group.

Patients and controls underwent the tests in a syncope clinic with autonomic laboratory facilities.

All participants had a normal electrocardiogram, were non-smokers and were not on medication, with the exception of birth control pills. All patients and controls were asked not to consume food or caloric beverages in the eight hours preceding the study or to drink coffee in the 24 hours before the study. Subjects with heart disease, diabetes or any other disease that could influence the results were excluded. The study protocol was approved by the Ethics Committee of Porto University Hospital and all patients gave their written informed consent.

In accordance with our protocol, each subject's assessment started at 10 am in a temperature-controlled room. After 15 min of bed rest, data were recorded for 10 min in the supine position, and subsequently during tilting in a head-up position at 70° with foot-board support for a maximum of 40 min. Continuous beat-by-beat recording (ECG and finger BP) was performed. The 10 min supine recording and the initial 10 min after tilting were used for comparison of autonomic, hemodynamic and neurohormonal parameters between groups.

Finger BP was obtained non-invasively with the Ohmeda 2300 device (Finapres®, Englewood, CO), which uses a photoplethysmographic technique. With this method, pressure waves from finger recordings with excellent correlation with intra-arterial pressure values4 are generated continuously. The ECG signal was acquired after careful preparation of the skin, to keep impedance <5 kΩ. A CM5-type lead was used to obtain a high-amplitude QRS complex in order to decrease R-wave recognition errors.5,8

Spectral analysis of HR (RR interval) and systolic blood pressure variability (SBVP) was performed with software developed in the Matlab® environment (The MathWorks, Inc., South Natick, MA), to provide a flexible analysis system. Spectral analysis was performed by the non-parametric Welch method.5,6,8

The spectrum was decomposed in the three classical bands: the high frequency component (HF), between 0.15 and 0.40 Hz equivalent (Hz eq)2; the low frequency component (LF), between 0.04 and 0.15 Hz eq; and the very low frequency component (VLF), between 0.01 and 0.04 Hz eq. All data were calculated in absolute values by the area under the curve of the respective spectra.

Assuming that variations in arterial pressure in the band centered around 0.10 Hz, obtained by spectral analysis of SBVP, represent rhythmic fluctuations of vasomotor activity mediated by the arterial baroreflex (Mayer waves), we calculated the spontaneous arterial baroreceptor gain (BRG) by spectral coherence.5 This band in the spectrum of heart rate variability (HRV) seems to correspond to baroreflex-mediated sympathetic and vagal adjustments.5,7,8 Baroreflex sensitivity was calculated from the modulus of the cross spectrum of RR interval and SBP for frequencies between 0.04 and 0.15 Hz.5,8

Stroke volume (SV), cardiac output and total peripheral resistance (TPR) were calculated by the method developed by Wesseling et al.9 because of its simplicity, low cost and non-invasive nature. This method analyzes the finger arterial pressure wave, as obtained by Finapres or Portapres devices or intra-arterial recordings, and calculates several hemodynamic parameters after applying the beat-to-beat model flow interpretation. An application of this three-element model has been published.10 Wesseling et al.9 described a good correlation, with an error of less than ±2%, between values obtained by this technique and thermodilution.

Ten ml of blood were collected in two different tubes: one containing EDTA and aprotinin (1.9 mg and 100 kIU/ml, respectively) for measurement of natriuretic peptides, and one heparinized tube containing 1.2 mg glutathione/ml for measurement of catecholamines.

Catecholamines were concentrated from plasma by liquid-liquid extraction and derivatized with the selective fluorescent agent 1,2-diphenylethylenediamine prior to chromatography. Optimal conditions for extraction, derivatization and chromatography have been investigated.11 Detection thresholds for this method are 0.3 pg for noradrenaline and adrenaline and 0.5 pg for dopamine. Atrial natriuretic peptide (ANP) was determined by radioimmunoassay (Nichols Institute) after extraction from plasma using Sep-Pak C18. BNP (brain natriuretic peptide) was measured by immunoradiometric assay (Shionoria), both without extraction.12 The intra-assay coefficient of variation of all methods used was in the range of <10%. All samples from each subject were analyzed in the same assay.

The samples were collected in supine rest and during the third minute of tilting.

Continuous variables with normal distribution were expressed as mean and standard deviation. The Kruskal-Wallis test was used to compare means of independent samples and the Wilcoxon signed rank test and the Mann-Whitney test were used to compare changes in paired samples and for pairwise comparisons. A p value <0.05 was accepted for rejection of the null hypothesis for all comparisons. The statistical analysis was performed with SPSS version 14.0 (SPSS Inc., Chicago, IL, USA).

SV was similar between groups in supine position (Table 1). SV decreased in response to tilting in all groups, particularly in OH (−37±18 ml, p=0.001) and POTS patients (−28±12 ml, p=0.008). TPR was similar in all four groups during supine rest and rose in all groups in response to tilt. However this rise (p=0.04) was lower in OH patients compared to other groups (p<0.01) and was greater in patients with POTS (+709±251 dyn.s.cm−5, p=0.008) than in other groups. Supine HR was similar in all groups. In response to head-up tilting HR did not rise in patients with OH (+8±7 bpm, NS) but rose sharply in patients with POTS (+42±10 bpm, p=0.001) compared to healthy controls (+13±6 bpm, p=0.008). Supine systolic blood pressure (SBP) was similar among groups. SBP decreased markedly in patients with OH (−41±11 mmHg, p=0.002) in response to head-up tilting but did not change in NM syncope patients (+8±4 mmHg, NS) or POTS patients (+2±3 mmHg, NS).

BRG was almost absent in OH patients (0.2±0.1 ms/mmHg in supine and 0.1±0.2 ms/mmHg in tilt position). At supine rest BRG was similar in POTS, NM syncope patients and healthy controls. After tilting, BRG in patients with POTS (−13.0±2.9 ms/mmHg, p=0.002) and NM (−11.6±7.0 ms/mmHg, p=0.007) decreased more than in healthy controls (−6.5±4.0 ms/mmHg, p=0.03), whereas vagal tone activity (HF) at different positions was similar in POTS, NM syncope and healthy controls (Tables 1–3). Low frequency of systolic blood pressure variability (LF-SBP) was almost absent in patients with OH (0.6±0.4 mmHg2 in supine and 1.0±0.7 mmHg2 in tilting position). It was similar in patients with NM syncope, POTS and in healthy controls in supine position. However in response to head-up tilt a large rise in LF-SBP occurred in patients with NM syncope (+11.6±5.4 mmHg2, p=0.004) and POTS patients (+11.6±3.9 mmHg2, p=0.003) as compared to healthy controls (+5.6±4.8 mmHg2 p=0.009).

Brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) did not change in response to tilt in any of the three patient groups or in controls. BNP was higher in patients with neurogenic OH (9.6±5.0 pmol/l, p=0.009) than in controls (1.5±1.1 pmol/l) and slightly lower in POTS patients (1.0±0.7 pmol/l, p=0.03). In patients with POTS, ANP in supine and tilt position was lower than in the other groups (p=0.03). Dopamine did not change with orthostatic stress in any group and levels in supine and tilt position were similar. As expected, patients with neurogenic OH had significant lower noradrenaline (NOR) in supine position (106±56 pg/ml, p=0.003) and a blunted, non-significant, response to orthostatic stress (+28±24 pg/ml, NS). POTS patients, NM patients and controls had a significant rise NOR after tilt. However patients with POTS compared to controls (197±109 pg/ml) had a more pronounced rise (340±102 pg/ml, p=0.03). Adrenaline was slightly elevated in patients with NM in supine position compared to the other groups and a large rise was observed during the first minutes of orthostatic stress and several minutes before the neurally mediated reflex started (+70±23 pg/ml, p=0.003). Adrenaline was similar in OH patients (22±5 pg/ml) and in controls (14±9 pg/ml) at supine rest but this parameter did not rise in response to tilting only (6±6 pg/ml in patients, NS, and 18±11 pg/ml in controls, p=0.04).

Tables 1–3 show detailed results.

The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

The authors declare that they have followed the protocols of their work center on the publication of patient data.

The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document.