Effects of short-term manipulation of serum FFA concentrations on

left ventricular energy metabolism and function in patients with heart failure; no association with circulating bio-markers of inflammation

Salerno A1MD, Fragasso G1MD, Esposito A23 MD, Canu T2 Mrs, Lattuada G4 PhD, Manzoni G4 MD, Del Maschio A23MD, Margonato A13 MD, De Cobelli F23 MD, Perseghin G45, MD

Supplemental material to Materials and Methods

Cardiac 31P-MR Spectroscopy.

Cardiac 31P-MR spectroscopy was performed within the Division of Diagnostic Radiology at rest using a 1.5T whole-body scanner (Gyroscan Intera Master 1.5 MR System; Philips Medical Systems, the Netherlands). 31P spectra were obtained by means of a 10-cm-diameter surface coil used for transmission and detection of radio frequency signals at the resonance frequency of 31P (at 1.5-T, 25.85 MHz). After appropriate positioning of the surface coil on the chest, localized homogeneity adjustment was performed using the body coil and ECG-triggering by optimizing the 1H-MR spectroscopy water signal. Afterwards the transmitter-receiver was switched without time delay to the 31P frequency and manual tuning and matching of the 31P surface coil was performed to adjust for different coil loading. The radio frequency level was adjusted to obtain a 180° pulse of 40 ms for the reference sample at the center of the 31P-surface coil. The acquisition of 31P-MR spectra was triggered to the R-wave of the ECG, with a trigger delay time of 200 ms and a recycle time of 3.6 s. ISIS volume selection in three dimensions (3D-ISIS) based on 192 averaged free induction decays was employed. The Volume of Interest was oriented avoiding inclusion of chest wall muscle and diaphragm muscle (typical volume size: 5 (caudo-cranial)x6x6 cm3). Acquisition time was 10 min. Adiabatic frequency-modulated hyperbolic secant pulses and adiabatic half-passage detection pulses were used to achieve inversion and excitation over the entire Volume of Interest.

Cardiac MR Imaging

MR imaging studies were performed with the above described scanner using an enhanced gradient system with a maximum gradient strength of 30 mT/m and a maximum gradient slew rate of 150 mT•m-1•sec-1. The Cardiac Research software patch (operating system 9) was used. The examination was performed using a 5-element cardiac phased-array coil (SENSE-cardiac) and retrospective ECG-triggering obtained with Vectorcardiogram system (VCG) and using standard MRI methodology as previously described.[13]

Calculations

31P-MR spectra were transferred to a remote SUN-SPARC workstation for analysis. The spectra were quantified automatically in the time domain, using Fitmasters as previously described (8,12) to obtain the PCr/ATP ratio. Low PCr/ATP ratio may be considered a typical metabolic feature of the hypertrophied and/or failing heart. As nicely reviewed by Neubaer S (7), the PCr/ATP ratio was reported to be reduced in heart failure, to be associated with parameters of systolic and diastolic LV function and with functional heart failure class, and was considered a better long-term prognostic indicator than LV ejection fraction. An estimate of the Signal-to-Noise-Ratio of each spectra was obtained from the relative Cramer-Rao standard deviation (rCRSD) calculated for the PCr/ATP, which is a commonly reported index of accuracy of the spectral quantification (14).

Random allocation to Study 1 or Study 2 first and duration of participation.

After the screening procedures, patients were allocated to undergo Study 1 or Study 2 first based on a pre-specified randomization schedule using the Software “Random Allocation” using a “only one block” random list (Study A or Study B first). The random list was available to a person working in the lab across the corridor and being part of another group of independent investigators.

The length of time between Study 1 and Study 2 was seven days for 9 subjects (the protocol was designed to be performed in two consecutive Wednesdays). One subject performed the Study fourteen days apart. One other subject performed the study twenty-eight days apart.

Supplemental on-line materials - Tables

Table 1. Anthropometric and laboratory features of each study subjects

Pat 1 / Pat 2 / Pat 3 / Pat 4 / Pat 5 / Pat 6 / Pat 7 / Pat 8 / Pat 9 / Pat 10 / Pat 11 / Mean±SD
Age (years) / 63 / 62 / 72 / 71 / 75 / 66 / 74 / 61 / 70 / 74 / 64 / 68±5
Body weight (kg) / 85 / 68 / 85 / 79 / 68 / 84 / 81 / 72 / 58 / 85 / 80 / 77±9
Height (cm) / 163 / 159 / 177 / 160 / 162 / 174 / 166 / 171 / 160 / 161 / 179 / 167±7
BMI (kg/m2) / 31.95 / 26.8 / 27.1 / 30.8 / 25.9 / 27.7 / 29.4 / 24.6 / 22.9 / 32.8 / 25.0 / 27.6±3.2
Waist (cm) / 105 / 88 / 95 / 96 / 84 / 90 / 98 / 81 / 81 / 101 / 93 / 92±8
Body fat (%) / 32.2 / 24.9 / 26.2 / 38.5 / 25.0 / 29.9 / 28.0 / 19.8 / 19.6 / 31.0 / 28.0 / 28±5
Systolic BP (mm Hg) / 140 / 128 / 124 / 135 / 134 / 140 / 126 / 129 / 141 / 140 / 133 / 134±6
Diastolic BP (mm Hg) / 80 / 75 / 75 / 64 / 65 / 80 / 61 / 87 / 82 / 82 / 85 / 75±9
Creatinine (µmol/L) / 87 / 66 / 98 / 113 / 140 / 69 / 128 / 69 / 65 / 99 / n.a / 94±279
Glucose (mmol/L) / 5.13 / 5.37 / 6.00 / 4.26 / 4.23 / 4.56 / 4.16 / 5.07 / 5.04 / 5.17 / 5.90 / 5.0±0.6
Total cholesterol (mmol/L) / 5.20 / 4.24 / 4.65 / 4.27 / 4.68 / 3.65 / 3.96 / 3.72 / 4.78 / 5.95 / n.a / 4.5±0.7
HDL cholesterol (mmol/L) / 1.40 / 1.01 / 1.32 / 1.29 / 1.58 / 1.11 / 0.70 / 1.29 / 1.29 / 1.71 / n.a / 1.24±0.29
Triglycerides (mmol/L) / 1.85 / 0.85 / 1.21 / 1.15 / 1.17 / 0.87 / 1.41 / 0.69 / 0.96 / 1.53 / 1.16 / 1.16±0.32
AST (U/L) / 26 / 15 / 29 / 31 / 17 / 18 / 20 / 24 / 19 / 22 / n.a. / 22±5
ALT (U/L) / 19 / 15 / 22 / 22 / 13 / 17 / 18 / 20 / 17 / 15 / n.a. / 17±3

Table 2. Parameters of diastolic function during Study 1 and Study 2.

Study 1 / Study 2
Basal / Study / Basal / Study / p-value
Early peak flow rate (mL/sec) / 220 ± 93 / 217 ± 109 / 222 ± 89 / 231 ± 104 / 0.584
Atrial peak flow rate (mL/sec) / 295 ± 133 / 302 ± 135 / 349 ± 138 / 354 ± 134 / 0.820
Early/Atrial peak flow rate ratio / 0.79 ± 0.91 / 0.69 ± 0.35 / 0.77 ± 0.48 / 0.78 ± 0.54 / 0.566
Deceleration time (msec) / 164 ± 77 / 191 ± 89 / 161 ± 69 / 192 ± 45 / 0.991

A two-factor repeated measures ANOVA was used to assess study 1 vs study 2 effect on the variables in the tables. To adjust for the potential effect of aging and body adiposity, the two-factor repeated measures ANOVA was performed using age and BMI as covariates but no significance was detected.

Legend to figure

In the left panel, the absolute values of the heart rate during in the baseline (gray box) and experimental (white box) conditions are summarized. No change was detected. On the right side the percent change from baseline is summarized (no difference between groups). Box plot shows data as median, confidence intervals and outliers.