Long-term effects of hepatocyte growth factor gene therapy in rat myocardial infarct model.

We investigated the long-term effects of human hepatocyte growth factor (HGF) gene therapy in a rat myocardial infarct model. Treatment adenovirus coexpressing the HGF therapeutic gene and the human sodium/iodide symporter (NIS) reporter gene or control adenovirus expressing the NIS gene alone were injected directly into the infarct border zone immediately after permanent coronary ligation in rats (n=6 each). A uniform disease state was confirmed in the acute phase in terms of impaired left ventricular (LV) function by cine magnetic resonance imaging (MRI), large infarct extent by (99m)Tc-tetrofosmin single-photon emission computed tomography (SPECT) and successful gene transfer and expression by (99m)TcO(4)(-) SPECT. After a 10-week follow-up, repeated cine MRI demonstrated no significant difference in the LV ejection fraction between the time points or groups, but a significantly increased end-diastolic volume from the acute to the chronic phase without a significant difference between the groups. Capillary density was significantly higher in the treatment group, whereas arteriole density remained unchanged. Two-photon excitation fluorescence microscopy revealed extremely thin capillaries (2-5 µm), and their irregular networks increased in the infarct border zone of the treated myocardium. Our results indicated that single HGF gene therapy alone induced an immature and irregular microvasculature.

Enhanced external counterpulsation improved myocardial perfusion and coronary flow reserve in patients with chronic stable angina; evaluation by(13)N-ammonia positron emission tomography.

AIMS: The mechanism by which enhanced external counterpulsation therapy exerts its beneficial effects on chronic and symptomatic stable angina is largely unknown. To clarify the mechanism of action of enhanced external counterpulsation, we used(13)N-ammonia positron emission tomography to evaluate myocardial perfusion. METHODS AND RESULTS: This was not a randomized controlled study. Eleven patients (eight male, age: 61.6+/-9.7) with angina pectoris underwent enhanced external counterpulsation therapy for 35 1 h sessions. They underwent a treadmill exercise test and(13)N-ammonia positron emission tomography, both at rest and with dipyridamole, before and after enhanced external counterpulsation therapy. Neurohumoral factors and nitric oxide were also evaluated. Myocardial perfusion increased at rest after therapy (0.69+/-0.27 to 0.85+/-0.47 ml x min(-1) x g(-1), P<0.05). In ischaemic regions, particularly the anterior region, myocardial perfusion at rest and with dipyridamole and coronary flow reserve improved significantly after therapy (at rest: 0.71+/-0.26 to 0.86+/-0.31;P<0.05, with dipyridamole: 1.26+/-0.65 to 1.84+/-0.94;P<0.02, coronary flow reserve: 1.75+/-0.24 to 2.08+/-0.28;P<0.04). Exercise time was prolonged and the time to 1-mm ST depression improved markedly (P<0.01). After therapy, nitric oxide levels increased (P<0.02) and neurohumoral factors decreased. CONCLUSIONS: Enhanced external counterpulsation therapy improved myocardial perfusion at rest and with dipyridamole and was associated with an increased exercise tolerance with(13)N-ammonia positron emission tomography and increased nitric oxide levels. These results suggest that one of the enhanced external counterpulsation mechanisms is development and recruitment of collateral vessels.

Embolic distribution through patent foramen ovale demonstrated by (99m)Tc-MAA brain SPECT after Valsalva radionuclide venography.

UNLABELLED: Cryptogenic stroke might relate to paradoxical embolism stemming from right-to-left shunt caused by patent foramen ovale (PFO). We performed radionuclide venography using the Valsalva maneuver, followed by (99m)Tc-macroaggregated albumin (MAA) brain SPECT to investigate the fate of emboli originating from the lower extremities. METHODS: Ten patients (9 men, 1 woman; mean age, 61 +/- 17 y) with PFO underwent radionuclide venography with and without the Valsalva maneuver on the whole-body image, followed by brain SPECT with (99m)Tc-MAA to determine the cortical uptake that would detect right-to-left shunt. After counts in each region of interest (ROI) were normalized by comparison with the averaged count, the distribution of MAA was compared with that of (99m)Tc-hexamethyl-propyleneamine oxime (HMPAO) brain SPECT by drawing ROIs on frontal, temporoparietal (anterior circulation territory), occipital, and cerebellar areas (posterior circulation territory). RESULTS: The thyroid on the whole-body scan was visualized after the Valsalva maneuver in 2 of the 10 patients. In 7 of 10 patients, 56 ROIs in the visualized cortical uptake showed that the distribution of MAA correlated well with that of HMPAO according to the equation: HMPAO = -71.21 + 1.71 x MAA, (r = 0.575, P < 0.01). The excess difference in the relative counts in the posterior over anterior circulation territory was 5.6% and 16.1% of the HMPAO and MAA values, respectively. CONCLUSION: Brain SPECT with (99m)Tc-MAA was more sensitive than thyroid visualization in detecting right-to-left shunt. The excess flow in the posterior cerebral circulation indicated an increased likelihood of cerebral emboli originating from the lower extremities and indicated that the flow difference between HMPAO and MAA probably resulted from poor linearization of HMPAO in the high-flow area.

Assessment of the effect of revascularization early after CABG using ECG-gated perfusion single-photon emission tomography.

When an arterial graft is used, reversible perfusion defects on single-photon emission tomography (SPET) perfusion images are occasionally observed early after coronary artery bypass graft surgery (CABG), owing to the restricted flow capacity. The purpose of this study was to determine whether the functional information obtained with electrocardiography (ECG)-gated perfusion SPET could be helpful in evaluating the effect of revascularization early after CABG. Twenty-three patients (18 men and 5 women, mean age 65+/-9 years) underwent stress/re-injection thallium-201 ECG-gated SPET before and 4 weeks after CABG (13 with exercise and 10 with dipyridamole). Patency of all grafts was confirmed by coronary angiography 1 month after CABG. Cardiac functional data including the left ventricular ejection fraction (LVEF) and the transient ischaemic dilatation (TID) ratio were analysed using a commercially available automated program. The conventional stress and re-injection tomograms were interpreted by means of a five-point scoring system in a nine-segment model. Stress-induced reversible 201Tl perfusion defects were present in 64% of the myocardial segments bypassed by patent arterial grafts, in contrast to 42% of the myocardial segments bypassed by patent venous grafts (chi2=7.8, P=0.005). Of the 23 patients, 12 showed improvement in summed ischaemic scores (group 1), while 11 had no change or deterioration (group 2), although all grafts were patent on postoperative catheterization. The TID ratio improved in both group 1 and group 2 before and after CABG (1.14+/-0.13 vs 0.99+/-0.07, P=0.001 and 1.09+/-0.07 vs 0.94+/-0.05, P=0.002, respectively). However, LVEF did not significantly improve in group 1 or group 2 after CABG (42.5%+/-9.9% vs 47.5%+/-11.8%, and 52.1%+/-7.5% vs 53.1%+/-5.9%, respectively). Perfusion imaging or LVEF assessment is of limited value early after CABG. The TID ratio obtained with ECG-gated perfusion SPET may be a useful marker to evaluate the effect of revascularization early after surgery.

Iodinated free fatty acid and 201T1 uptake in chronically hypoperfused myocardium: histologic correlation study.

UNLABELLED: 123I-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) is a tracer for the evaluation of ischemic heart disease. The purpose of this study was to assess the relationship between 1231-BMIPP uptake and myocardial fibrosis. METHODS: Fifteen patients who underwent cardiac surgery were examined by imaging with 201TI and 123I-BMIPP, and histologic specimens were taken during surgery. The relative uptake of 201TI (%TI) and that of 123I-BMIPP (%BMIPP) were calculated. The percentage of fibrosis (%fibrosis) was analyzed with the specimen. RESULTS: %TI correlated strongly with %fibrosis (r = -0.94; P < 0.001). %BMIPP also correlated significantly with %fibrosis (r = -0.88; P < 0.001), but the change in %BMIPP looked biphasic. In the category of only mild fibrosis, %BMIPP showed a steep decrease. 123I-BMIPP-201TI mismatch was found only for fibrosis <20%. CONCLUSION: 123I-BMIPP gave specific information about metabolic changes that occurred in ischemic myocardium without severe fibrotic changes.

Simultaneous assessment of myocardial free fatty acid utilization and left ventricular function using 123I-BMIPP-gated SPECT.

UNLABELLED: This study was designed to evaluate the methodological feasibility of 123I-labeled beta-methyl-p-iodophenyl-pentadecanoic acid (BMIPP)-gated SPECT to assess regional and global left ventricular (LV) function in comparison with 99mTc-sestamibi (methoxyisobutyl isonitrile [MIBI])-gated SPECT and first-pass radionuclide angiography (FPRNA). METHODS: Forty-four patients with stable coronary artery disease underwent rest BMIPP-gated SPECT (111 MBq, 60 s/step) and rest MIBI-gated SPECT (600 MBq, 40 s/step) within a week. From both gated SPECT studies, regional defect scores (DS), wall motion scores (WMS) and wall-thickening scores (WTS) were evaluated visually using 4-point scales for nine segments, and LV ejection fraction (EF) (%) was automatically calculated using Quantitative Gated SPECT (QGS) software. FPRNA was also performed on injection of MIBI. RESULTS: Exact agreement between the two gated SPECT studies was 84.1% (kappa = 0.706, r = 0.907, P < 0.0001) in WMS and 87.1% (kappa = 0.662, r = 0.884, P < 0.0001) in WTS. LVEF obtained from BMIPP-gated SPECT linearly correlated with those from MIBI-gated SPECT (y = -0.27 + 0.944x, r = 0.948, SEE = 5.00, P < 0.0001) and FPRNA (y = -7.32 + 1.042x, r = 0.919, SEE = 6.19, P < 0.0001). Even in 21 patients with mismatch segments (BMIPP DS > MIBI DS), agreement was considered to be acceptable in WMS (81.5%, kappa = 0.707, r = 0.853, P < 0.0001) and in WTS (76.7%, kappa = 0.526, r = 0.754, P < 0.0001), and correlation in LVEF remained good between BMIPP-gated SPECT and MIBI-gated SPECT (y = -1.24 + 0.955x, r = 0.938, SEE = 6.25, P < 0.0001) or FPRNA (y = -6.03 + 1.024x, r = 0.913, SEE = 7.38, P < 0.0001). CONCLUSION: BMIPP-gated SPECT can evaluate regional and global LV function with the QGS software. Therefore, BMIPP-gated SPECT offers the opportunity for simultaneous assessment of myocardial free fatty acid utilization and LV function.

Use of technetium-99m sestamibi ECG-gated single-photon emission tomography for the evaluation of left ventricular function following coronary artery bypass graft: comparison with three-dimensional magnetic resonance imaging.

In patients who had undergone cardiac surgery (coronary artery bypass graft) and whose hearts showed abnormal movement during the cardiac cycle, we studied the accuracy of functional assessment using ECG-gated single-photon emission tomography (SPET) and the automated software developed by Germano et al. by comparing the findings with magnetic resonance (MR) images acquired three-dimensionally. Sixteen patients who had undergone cardiac surgery underwent 99mTc-sestamibi gated SPET (MIBI-g-SPET) and MRI on the same day. Left ventricular end-diastolic and end-systolic volumes (EDV, ESV) and ejection fraction (LVEF) were measured using MIBI-g-SPET and the aforementioned algorithm. Regional wall thickening was assessed using a four-point scale on MIBI-g-SPET and cine MRI. There was a good correlation between MIBI-g-SPET and MRI in respect of EDV (r=0.89), ESV (r=0.93) and LVEF (r=0.89). A high degree of agreement was found between the wall thickening scores obtained by MIBI-g-SPET and MRI in total segments (kappa=0.62) and in septal segments (kappa=0.67). It is concluded that ECG-gated perfusion SPET can provide regional and global functional information, including absolute volumes, in patients following cardiac surgery.

Assessment of regional and global left ventricular function by reinjection T1-201 and rest Tc-99m sestamibi ECG-gated SPECT: comparison with three-dimensional magnetic resonance imaging.

OBJECTIVES: The purpose of this study was to test the ability of reinjection thallium-201 and rest technetium-99m sestamibi ECG (electrocardiographic)-gated SPECT (i.e., reinjection-g-SPECT [single-photon emission computed tomography] and MIBI-g-SPECT) to determine regional and global functional parameters. BACKGROUND: The ECG-gated perfusion SPECT was reported to provide accurate left ventricular ejection fraction (LVEF) using an automated algorithm. We hypothesized that other various functional data may be obtained using reinjection-g-SPECT and MIBI-g-SPECT. METHODS: Reinjection-g-SPECT, MIBI-g-SPECT, and three-dimensional magnetic resonance imaging (3DMRI) were conducted in 20 patients with coronary artery disease. Regional wall motion (RWM) and wall thickening (RWT) were analyzed using semiquantitative visual scoring by each g-SPECT and 3DMRI. The left ventricular end-systolic and end-diastolic volumes (EDV, ESV) and LVEF estimated by reinjection- and MIBI-g-SPECT were compared with the results of 3DMRI. RESULTS: A high degree of agreement in RWM and RWT assessment was observed between each g-SPECT and 3DMRI (kappa >.70, p < .001). The LVEF values by reinjection- and MIBI-g-SPECT correlated and agreed well with those by 3DMRI (reinjection: r = .92, SEE = 5.9%, SD of differences = 5.7%; sestamibi: r = .94, SEE = 4.4%, SD of differences = 5.1%). The same also pertained to EDV (reinjection: r = .85, SEE = 18.7 ml, SD of differences = 18.4 ml; sestamibi: r = .92, SEE = 13.1 ml, SD of differences = 13.0 ml) and ESV (reinjection: r = .94, SEE = 10.3 ml, SD of differences = 10.3 ml; sestamibi: r = .97, SEE = 6.7 ml [p < .05 vs. reinjection by F test], SD of differences = 6.6 ml [p < .05 vs. reinjection by F test]). CONCLUSIONS: Reinjection- and MIBI-g-SPECT provide clinically satisfactory various functional data. These functional data in combination with the perfusion information will improve diagnostic and prognostic accuracy without an increase in cost or the radiation dose to the patients.

An ultrashort 1-day protocol of Tc-99m tetrofosmin.

PURPOSE: This study describes a new 1-day protocol with Tc-99m tetrofosmin that requires only 100 minutes to obtain both stress and resting cardiac images by using a double-injection and subtraction method. METHODS: This procedure was performed in 48 consecutive patients. Rest-rest double injections were performed in eight patients (five men, three women; mean age, 69 +/- 9.8 years ) to evaluate count and image reproducibility (subprotocol A), and stress-rest and additional resting perfusion images (true rest) were done on a different day in 11 patients (five men, six women; mean age, 63 +/- 5.9 years) to confirm the validity of the new protocol (subprotocol B). RESULTS: Image quality scores of the resting image were excellent (35 of 48, or 72.9%), good (7 of 48, or 14.6%), fair (3 of 48, or 6.3%), and poor (3 of 48, or 6.3%). The scintigraphic findings with the new protocol corresponded closely with those of angiography in 26 of 34 cases (76.5%), with a tendency for underestimation (in 5 of 34 cases, or 14.7%) rather than overestimation (in 3 of 34 cases, or 8.8%). In subprotocol A, count reproducibility between the two resting images was excellent (r = 0.95; P < 0.0001); and in subprotocol B, the early-rest images were concordant visually and quantitatively with the true rest images (r = 0.89, P < 0.0001). CONCLUSION: Although there are some limitations, this protocol can be used as a routine stress-rest protocol.

Improvement of regional myocardial and coronary blood flow reserve in a patient treated with enhanced external counterpulsation: evaluation by nitrogen-13 ammonia PET.

Enhanced external counterpulsation (EECP) is a noninvasive treatment for chronic stable angina, which works by recruiting and developing the coronary collateral vessels. Coronary perfusion and coronary flow reserve (CFR) were evaluated by nitrogen-13 (13N) ammonia positron emission tomography (PET) in a patient who had undergone EECP. The patient, who had 3-vessel coronary artery disease, required a percutaneous transluminal coronary angioplasty (PTCA) for the right coronary artery. The PTCA was successful, but 6 months later he again felt chest oppression. The coronary angiography showed re-stenosis at the PTCA site, and other progressive coronary stenosis. The patient was again treated with EECP for 35 h. The 13N-ammonia PET was performed both at baseline and during dipyridamole provocation, before and after EECP treatment. Coronary perfusion of each myocardial wall increased at the baseline (anterior: 0.52-0.75; septal: 0.48-0.66; lateral: 0.61-0.68; inferior: 0.46-0.57 ml min(-1) g(-1), and the CFRs in the septal and inferior walls (septal: 2.07-2.15; inferior: 1.99-2.06) also increased after the treatment. Thus, the EECP treatment improved both coronary perfusion at baseline and CFR, which suggests that it may be one of the choices for treatment of angina.

Abnormality of myocardial oxidative metabolism in noninsulin-dependent diabetes mellitus.

UNLABELLED: The purpose of this study was to evaluate oxidative metabolism and its response by dobutamine in patients with noninsulin-dependent diabetes mellitus (NIDDM) using 11C-acetate PET. METHODS: We studied 16 patients with NIDDM (9 men, 7 women; mean age 53.7 +/- 12.8 yr) and 6 healthy male control subjects (mean age 41.8 +/- 17.2 yr). None of them had an abnormality on stress-perfusion SPECT. The 11C-acetate clearances (Kmono) were compared regionally for five myocardial segments in all subjects at rest and during low-dose dobutamine stress in 13 patients (8 patients with NIDDM, age 51.9 +/- 13.6 yr; 5 healthy male control subjects, age 45.6 +/- 16.3 yr). Correlation between regional Kmono and rate-pressure product (RPP) was also studied. RESULTS: At rest, the clearance of 11C-acetate was slightly heterogeneous for both patients with NIDDM and healthy control subjects, with smaller values in the apex and inferior wall in both groups. The difference became significant during dobutamine stress in the patients. The RPP-to-Kmono (average for five segments) ratio at rest was slightly smaller in the patients (1042.7 +/- 559.1 x 0.01) than in the healthy control subjects (1391.4 +/- 209.6 x 0.01, not significant), and those during dobutamine stress were almost the same in the two groups (1457.3 +/- 737.4 x 0.01 and 1486.0 +/- 211.8 x 0.01, respectively). A significant correlation was seen between regional Kmono and RPP in every segment in the healthy control subjects (average; r = 0.89; p < 0.01), whereas more scattered correlation with greater regional variation was observed in the patients (average; r = 0.31; p value was not significant). CONCLUSION: Patients with NIDDM showed slight regional heterogeneity in myocardial oxidative metabolism. They also had more scattered correlation between myocardial oxidative metabolism and cardiac work (RPP) than healthy control subjects, with the smallest correlation coefficient observed in the inferior wall. These findings may help the understanding of dynamics in myocardial oxidative metabolism of NIDDM hearts.

First-pass radionuclide angiography using iodine-123 myocardial tracers and a multicrystal gamma camera.

UNLABELLED: The purpose of this study was to validate the accuracy of the assessment of ventricular function by first-pass radionuclide angiography (FPRNA) with 123I myocardial tracers and a multicrystal gamma camera. METHODS: Left ventricular ejection fraction (LVEF) and right ventricular ejection fraction were measured in 69 patients by FPRNA using 123I myocardial tracers (126 +/- 7 MBq) and 99mTc tracers (541 +/- 141 MBq) on a multicrystal gamma camera with a high-sensitivity collimator. For 44 patients, ejection fraction values measured by 123I-FPRNA were compared to those estimated by equilibrium radionuclide angiography (ERNA). Visual wall-motion analysis was also performed to judge clinical acceptability of 123I-FPRNA images for identification of wall-motion abnormality. RESULTS: Mean LVEFs (%) estimated by 123I-FPRNA and by 99mTc-FPRNA were 49.6 +/- 13.6 and 49.1 +/- 14.1, respectively (nonsignificant p value). An excellent correlation was found between LVEFs estimated by 123I-FPRNA and 99mTc-FPRNA (r = 0.96, s.e.e. = 1.9%). Values of LVEF measured by 123I-FPRNA also demonstrated excellent correlation with those measured by ERNA (r = 0.95, s.e.e. = 2.2%). A good correlation was also noted between right ventricular ejection fractions measured by 123I-FPRNA and 99mTc-FPRNA (r = 0.72, s.e.e. = 4.0%). The Spearman rank correlation coefficient between 123I-FPRNA and ERNA wall-motion scores was 0.87 (n = 135, p < 0.001). CONCLUSION: Resting ventricular function can be reliably measured with 123I-FPRNA in combination with a multicrystal gamma camera. This indicates that the assessment of ventricular function is feasible in conjunction with 123I myocardial imaging without an increase in cost or radiation dose to patients.

Impairment of BMIPP uptake precedes abnormalities in oxygen and glucose metabolism in hypertrophic cardiomyopathy.

UNLABELLED: Impairment of fatty acid uptake is shown to precede myocardial perfusion abnormality using 123I-labeled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) in an experimental model of hypertrophic cardiomyopathy (HCM) and in human studies. We have recently demonstrated that abnormalities of both glucose and oxidative metabolism precede the reduction of blood flow in HCM. The main purposes of this study were to assess the frequency of abnormal findings in FDG uptake, BMIPP uptake and oxygen metabolism and to clarify the relationship among these metabolic parameters by using PET and SPECT. METHODS: Twenty-eight subjects with HCM underwent FDG- and acetate-PET and thallium- and BMIPP-SPECT studies at rest, respectively. After correcting for partial volume effect, real percentages of FDG and BMIPP uptake were calculated. In addition, the clearance rate constant (K mono) of acetate was measured and normalized (%) to estimate the oxygen metabolism. RESULTS: There were various metabolic abnormalities observed in patients with HCM. BMIPP uptake was often impaired without significant reduction of K mono values or FDG uptake. Thus, abnormality of BMIPP uptake was more frequently observed than that for FDG uptake or K mono values (p < 0.0001, respectively). FDG uptake was relatively maintained even in the segments with reduced K mono values and reduced BMIPP uptake. CONCLUSION: HCM shows a variety of metabolic patterns; however, the results of our study suggest that reduction of BMIPP uptake appears to be the most sensitive indicator of metabolic abnormalities followed by reduction of oxidative metabolism in patients with HCM.

Recent advances in nuclear cardiology in the study of coronary artery disease.

A variety of new radiopharmaceutical agents have been introduced to probe myocardial function in vivo. This review will introduce these new techniques which have recently been available in Japan. Tc-99m perfusion imaging agents provide excellent myocardial perfusion images which may enhance diagnostic accuracy in the study of coronary artery disease. In addition, greater photon flux from the tracer permits simultaneous assessment of regional perfusion and function with use of first-pass angiography or ECG-gated acquisition. Positron emission tomography enables metabolic assessment in vivo. Preserved FDG uptake indicates ischemic but viable myocardium which is likely to improve regional dysfunction after revascularization. In addition, FDG-PET seems to be valuable for selecting a high risk subgroup. Recently I-123 BMIPP, a branched fatty acid analog, has been clinically available in Japan. Less uptake of BMIPP than thallium is often observed in the ischemic myocardium. Such perfusion metabolic mismatch which seems to be similarly observed in FDG-PET is identified in the stunned or hibernating myocardium with regional dysfunction. Both of them are likely to recover afterwards. Severe ischemia is identified as reduced BMIPP uptake at rest, suggesting its role as an ischemic memory imaging. I-123 MIBG uptake in the myocardium reflects adrenergic neuronal function in vivo. In the study of coronary artery disease, neuronal denervation is often observed around the infarcted myocardium and post ischemic region as well. More importantly, reduced MIBG uptake in these patients can identify high risk for ventricular arrhythmias and assess severity of congestive heart failure. These new techniques will provide insights into new pathological states in the ischemic heart disease and enable to select optimal treatment in these patients.

Simultaneous determination of plasma phenytoin and its primary hydroxylated metabolites in carbon tetrachloride-intoxicated rats by high-performance liquid chromatography.

We have developed a simple and sensitive method for the simultaneous determination of phenytoin (PHT), 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in rat plasma by high-performance liquid chromatography. The three substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 250 mm x 4.6 mm I.D.) using acetonitrile-0.008 M NaH2PO4 (pH 6) (35:65, v/v) as a mobile phase at a flow-rate of 0.8 ml/min. Absorbance was monitored at 215 nm. The quantification limit was 50 ng/ml for each of PHT, m-HPPH and p-HPPH. The mean recoveries for DPH, m-HPPH and p-HPPH from plasma were 95.6 +/- 3.6, 94.5 +/- 4.2 and 98.6 +/- 2.9%, respectively.