MAGMA (1993) 1, 55-60

31p-NMR spectroscopy of human blood and serum: first results from volunteers and patients with

congestive heart failure, diabetes mellitus and hyperlipidaemia

Michael Horn*, Stefan Neubauer, Michael Bomhard, Marcus Kadgien, Klaus Schnackerz and Georg Ertl

Departments of Medicine and Physiological Chemistry, University of W~irzburg, Germany

31P-containing metabolites in human blood, serum and erythrocytes were measured or calculated. Phosphodiesters were found in serum, but not in erythrocytes. 2,3-diphosphoglycerate and 2,3-diphosphoglycerate/ATP ratios were increased in patients with congestive heart failure (2,3-diphosphoglycerate by 13% in mild to moderate, 31% in severe congestive heart failure, 2,3-diphosphoglycerate/ATP ratio by 9% in mild to moderate, 38% in severe congestive heart failure); phosphodiesters were increased in diabetes mellitus (by 26%) and even more so in hyper- lipidaemia (by 57%). Changes of blood 3~p compounds with disease states may have diagnostic potential and should be recognized for correction of organ spectra.

Keywords: congestive heart failure, diabetes mellitus, hyperlipidaemia, 31p NMR spectroscopy, blood, serum.

INTRODUCTION METHODS AND MATERIALS

The presence of blood inevitably contributes signal to in-vivo 31p-NMR spectra of human organs to a greater or lesser extent. Thus, definition of absolute and relative (ratios) amounts of 3~p_containing metabolites in human blood and serum is important. Blood correction of organ spectra has been previously applied based on an estimation of the 2,3-diphospho- glycerate/ATP ratio in blood from healthy individuals [1-3]. However, thorough quantitative analysis of 31p-compounds in blood and serum from volunteers and patients has been unavailable. Based on such information, appropriate blood correction of 3~p_ s~ectra from organs should be feasible. In addition, 3 P-NMR of blood and serum may by itself bear diagnostic and, possibly, prognostic potential for a variety of disease states. In this work, we characterize 31p-NMR blood spectra from volunteers and patients with congestive heart failure, diabetes mellitus and hyperlipidaemia.

* Address for correspondence: Medizinische Universitdtsklinik, Josef-Schneider-Str. 2, 8700 W~rzburg, Germany. Received 4 December 1992; accepted 27 February 1993.

Blood was drawn by venous puncture between 7.30 and 8.00 AM before patients had breakfast. Coagula- tion was inhibited with Na4EDTA. 2.5 ml of blood was used to obtain 31p-spectra; serum was obtained by centrifugation of 10 ml of additional blood. Samples were immediately placed on ice and transferred to the NMR unit within 30m in. Preliminary testing for stability of 31p-compounds in blood showed un- changed signals within 12h at 0°C or within 4h at 37°C. After rewarming to 37°C, one 31p-NMR spectrum was obtained (1032 acquisitions, 42min, interpulse delay 2.1 s, pulse angle 45°). Then, another 3~p-NMR spectrum of serum from the same subject was acquired using identical pulse parameters.

31P-NMR spectra were obtained on a Bruker AM 300 SWB (7.05 T magnet, Aspect 3000 computer) using a standard 10 mm multinuclear probe. Before acquiring spectra, the homogenity of the B0-field was optimized by shimming on the 2D-lock level. Data are corrected for partial saturation (1.03 for 2,3-DPG, 1.00 for PDE, y-P-, a-P- and ~-P-ATP). Saturation factors were obtained from fully relaxed spectra (TR = 24 s,

0968-5243 © 1993 Chapman &Hall

56 M HORN et al.

= 45°). The difference in the areas of the three P-ATP-signals is caused by some overlap wi th other 31p-compounds, e.g. ADP, NADP and other d iphosphoesters . However , the 7-P-ATP has the best S/N due to only one P-P-coupl ing (with fl-P-ATP). In addition, y-P-ATP is d o s e d to the excitation f requency thus no effects of insufficient excitation cause loss of signal.

Absolute quanti tat ion of compounds was achieved by compar ing resonance areas with a O,O ' -d imethyl - me thy lphosphona te (3.64 x 10 -s mol) s tandard (6 = 38.5 p p m versus 85% H3PO4) which was placed in a D20-conta in ing concentric compar tment added to the NMR tube.

Concentra t ions of 2,3-DPG, PDE and ATP in erythrocytes were calculated according to:

2,3-DPGe = 2,3-DPGB/(1 - Hkt) (1)

7-P-ATP e = 7-P-ATPb/(1 - Hkt) (2)

PDEe = (PDEb - (1 - Hkt) X PDEs)/Hkt (3)

where Hkt = blood hematocri t , e, b, s = concen- tration of compounds in erythrocytes , blood and s e m i .

Cell count and rout ine se rum chemistry were deter- mined from simultaneously d rawn blood samples (hematocrit (Hkt), erythrocytes (Coulter), glucose,

triglycerides (GPO-PAP method, Boehringer Mann- heim Diagnostica) and cholesterol (CHOD-PAP method , Boehringer Mannhe im Diagnostica)).

The free ATP-concentrat ion was calculated according to the me thod [4-6] of Gupta et aI.

Patient groups

Blood specimen were obtained from heal thy volun- teers (VOL; n = 13, age = 25.3 _+ 1.2) and from three groups of patients.

Patients with chronic congestive hear t failure (CHF) according to the N e w York Hear t Association (NYHA) classification (class II or III = mild or moderate , CHF~, n = 9, age = 69.3 + 4.9; class IV = severe, CHF s, n = 5, age = 59.0 + 9.3). All patients had evidence of left ventricular dysfunct ion u p o n echo- cardiography or radio-contrast ventr iculography. All 14 patients were on diuretics, eight had digitalis, ten an ACE-inhibitor, five Warfarin and two an anti- arrhythmic agent.

Patients with diabetes mellitus (DM; n = 5, age = 63.4 _+ 6.3, one pat ient with type 2a, four patients with type 2b) were all insu l in-dependent for 12.7 _+ 3.9 years. These patients had no evidence of hear t disease, a se rum creatinine of 0.94 + 0.08, and no excessive hyperl ipidaemia (Table 1). Two had per ipheral occlusive disease.

T a b l e 1. 31p-NMR spect roscop ic data and results of cell count and serum chemist ry .

VOL CHFm CHF~ HL DM

n 13 9 5 5 Age 25.3 _+ 1.2 69.3 _+ 4.9 59.0 _+ 1.2 53.4 + 7.3 7-P-ATPb (mM) 1.68 +- 0.28 1.84 _+ 0.29 1.61 + 0.13 1.90 _+ 0.52 ~-P-ATPb (mM) 1.45 _+ 0.25 1.39 _+ 0.09 1.35 + 0.13 1.52 _+ 1.64 2,3 -DPG/ /~ -P-ATPb 8.85 _+ 0.45 10 .13 + 0 .75 12 .65 + 2.48 a 8,47 _+ 0.60 PDE/y-P-ATPb 1.36 + 0,13 1.26 _+ 0.21 1.12 -+ 0.20 2.32 -+ 0.37 a PDE///-P-ATPb 1.58 _+ 0.14 1.47 _+ 0.20 1.35 _+ 0.24 2.33 _+ 0.19 a Pi s (mM) 1.22 + 0.12 1.00 _+ 0.10 0.83 +- 0.17 a 1.83 _+ 1.61 a PDE s (mM) 4.69 _+ 0,69 3.74 + 0.46 4.14 _+ 0.39 8.58 _+ 4.27 a 2,3-DPGe b (raM) 28.25 + 3,03 36.28 _+ 1.77" 36.49 _+ 0.29 27.87 + 2.44 7-P-ATPe b (raM) 3.85 _+ 0.54 4.72 _+ 0.48 3.69 --+ 0.29 4.65 + 1.13 ~-P -ATPe b (mM) 5.00 _+ 0.58 5.83 _+ 0,47 5.40 _+ 0.32 5.00 _+ 0.27 /~-P-ATPe b (mM) 3.28 _+ 0,46 3,70 _+ 0,27 3.10 -+ 0.29 3.79 _+ 0.34 2,3-DPG/y-P-ATPe b 8.82_+ 1.02 8.18-+0.74 10.38+ 1.70 8.11 _+ 1.64 2,3-DPG//~-P-ATPe b 10.24 _+ 1.01 10.14 _+ 0.76 12.66 _+ 2.48 8.47 _+ 0.81 Hkt (%) 44.87_+0.96 38.13-+2.13 a 43.82-+1.87 40 .16+2 ,43 ~ Glucose (mg%) 91.08 + 4.39 108.86 + 18.7 132.8 + 36,5 ~ 156.4 + 30,2 a T r i g l y c e r i d e s ( m g % ) 119.9_+ 15,2 96.0+_ 14.1 106.6-+ 18.4 635.6_+ 110.5 a Cholestero l (mg%) 195.6 -+ 8.9 179.7 -+ 17.9 182.6 _+ 25.5 345.6 _+ 27.5" Free ATP (%) 20.3 _+ 5,5 14.7 _+ 1,4 13.9 _+ 2.6 12.6 _+ 2.5

5 63.4 + 6,3 1.44 _+ 0.22 1.13_+0.17 9.95 _+ 0,28 3.30 + 1.24 a 4.17 _+ 1.55 a 1.57 _+ 0.14 5.69 _+ 0,89

28.89 _+ 1.82 4.10-+0.19 5.09 + 0.32 3.12_+0.17 7.71 -+ 0.30 9.86 + 0.24

35.74 -+ 4.37 a 178.8 _+ 22.9 a 217.0 -+ 18.4 a 225.6 _+ 33.1

13.0_+ 1.5

Results are mean + SE. a p < 0.05 versus VOL, unpaired one-tailed t-test. b Calculated e, b, s = concentration of compounds in erythrocytes, blood and serum.

MAGMA (1993) 1(2)

3~p-NMR-SPECTROSCOPY OF HUMAN BLOOD AND SERUM 57

Patients with hyperlipidaemia (HL; n = 5, age = 53.4 + 7.3) had no evidence of heart, kidney or liver disease. Classification according to Fredrickson showed four patients with type IV and one patient with type V. None of them was treated with lipid- lowering agents. Patients suffering from more than one of the disease states listed above were excluded.

STATISTICAL ANALYSIS

For each group of patients, data for each parameter obtained were compared with those from volunteers using the unpaired, one-tailed t-test. Calculations were aided by the StatView SE + Graphics, Statistics Utility (Abacus Concepts, Berkeley, CA, USA). All data are mean + s~. p-values of K 0.05 were considered significant.

RESULTS

In all cases, ~P-NMR spectra of blood showed resonances for 2,3-diphosphoglycerate (2,3-DPG), phosphodiesters (PDEB), y-, ¢¢- and /~-P-ATP. In serum, peaks for inorganic phosphate (Pis) and PDE (PDE~) were visible. T~pical examples are shown in Figs 1 and 2. Other P-metabolites of blood, e.g. glucose-6-phosphate, ADP, AMP and other products of ATP degradation, are invisible due to low concen- tration. The signal of Pi in blood is unresolvable due to overlap with the signals of 2,3-DPG. Based on Eq. 3, erythrocytes were calculated to contain all 2,3-DPG and ATP, but no PDE. The erythrocytes of several samples were spun down and ~P-NMR-spectra were obtained. In these spectra no PDE was visible and the calculation following Eq. 3 was proved. There was no change in concentrations of free ATP among the various groups (Table 1).

V O L U m ~

;~,3-DPG

DIABETES MELLIIUS

DMMP PDIE o.ATP DMMP

CONGeStIVE I~JLRT FAHJUIq~ (NYIIA IV) H'YPE~n'n)m~A

2,3-DPG

OMMP

~ATP

' L p-ATP

2,S-DPG

PDE

IDMIIIIP y-ATP a,A'rP

Fig. 1. Examples of 31p-NMR spectra of blood from a volunteer, a patient with congestive heart failure, one with diabetes mellitus and one with hyperlipidemia. 1032 acquisitions, 42 rain, interpulse delay 2.1 s, pulse angle 45 °. DMMP, O,O'- dimethylmethylphosphonate; 2,3-DPG, 2,3-diphosphoglycerate; PDE, phosphodiesters; 7'-, ~-, /~-phosphorus atoms of ATP.

M A G M A (1993) 1(2)

58 M HORN et a l.

VOLUNTEER D I A B ~ MELLITUS

POE

DMMP

POE

CONGF.hW[VE I~ART FK1LLrp~ ~ IV)

PDE DINMP

PD£

Fig. 2. Examples of 31p-NMR spectra of serum from a volunteer, a patient with congestive heart failure, one with diabetes mellitus and one with hyperlipidemia. 1032 acquisitions, 42 min, interpulse delay 2.1 s, pulse angle 45 °. DMMP, O,O'- dimethylmethylphosphonate; Pi, inorganic phosphate; PDE, phosphodiesters.

Absolute and relative amounts of 31p-compounds for the various groups are shown in Figs 3 and 4 and in Table 1. Although the majority of parameters were similar among groups, significant alterations of 31p_ NMR blood spectra were detectable. Both 2,3-DPG and the 2,3-DPG/ATP ratio increased in patients with heart failure, and the magnitude of this increase was related to the severity of hear failure (Fig. 3, p < 0.05 CHFs versus VOL). PDE levels in both blood and serum (Fig. 4) as well as PDE/ATP ratios (Table 1) were markedly elevated (1.57-fold for PDE, 1.71-fold for PDE/ATP ratio, both p < 0.05 HL versus VOL) in patients with hyperlipidemia. Similarly, in patients with diabetes mellitus, the PDE/ATP ratio (Table 1) was significantly increased (2.43-fold, p < 0.05 DM versus VOL).

DISCUSSION

In this preliminary report, absolute and relative amounts of 31P-compounds in human blood, serum and erythrocytes were measured or calculated. The

MAGMA (1993) 1(2)

2O

g

d

5

0 VOL CHFm CHFs DM HYP

14

12

a,. 10

<_.8

4

2

0 VOL CHFrn CHFs DM HYP

Fig. 3. (a) Concentration (mM) of blood 2,3-DPG for volunteers and the various patient groups (*p < 0.05), (b) Blood 2,3-DPG/ATP ratios for volunteers and the various patient groups (*p < 0.05).

31p-NMR-SPECTROSCOPY OF HUMAN BLOOD AND SERUM 59

12

10 [ ] PDEb [ ] PDEs

"-" 8

~ 4 ~ 6 ~ ~ ~ 2 *

0 VOL C H F m CHFs DM HYP

Fig. 4. Concentration (mM) of PDE in blood and serum for volunteers and the various patient groups (*p < 0.05).

PDE resonance of blood spectra was found to arise exclusively from serum, not from erythrocytes. This does not exclude the possibility of some immobile, NMR-invisible PDE in erythrocyte membranes, however.

Although cold storage of packed erythrocytes of rabbit [7] is reported to cause depletion of ATP over 72 h, we found stability of blood for 4 h at 37 °C and for 12 h at 0 °C. In preliminary experiments, freezing and thawing of blood led to 20-30% loss of ATP.

The relative amount of free ATP is dependent on the availability of oxygen [4]. In venous blood, we found no difference among the various disease states. Stability of Mg-ATP in blood from venous puncture has been previously reported even for extended storage at low temperatures [7], and is due to low metabolic rates of erythrocytes depending entirely on glycolysis which can continue with glucose being present in serum. Free ATP levels might vary, how- ever, if arterial blood sealed for oxygen diffusion was examined.

In congestive heart failure, we detected increased 2,3-DPGB levels and 2,3-DPG/y-P-ATPB and 2,3-DPG/ /~-P-ATPb ratios. Furthermore, the extent of 2,3-DPG and 2,3-DPG/ATP elevation was related to the severity of heart failure. One mechanism responsible for this effect is most likely mild chronic arterial hypoxemia due to decreased cardiac output. Increased 2,3-DPG levels in blood during mild hypoxemia have been reported previously [9]. Follow-up studies of a larger number of patients should allow us to examine the correlation between cardiac output, arterial oxygen saturation and 2,3-DPG blood levels. For the present work, we did not measure the arterial oxygen saturation (sO2art). However, clinical experience shows that the

SO2art does not change until patients with DCM are terminally ill, i.e. have a severely reduced cardiac output. Thus, we would not expect major changes of sO2art, although small changes might occur.

We found higher 2,3-DPG-levels in blood of volun- teers than a calculation based on the values of Bartlett [9] who measured levels of various phosphorus com- pounds in erythrocytes by labelling with radioactive 2p-compounds. Labotka et al. [10], however,

compared concentrations for various 31p-metabolites of blood (e.g. ATP and 2,3-DPG) by NMR and enzy- matic methods and report coincidence of both methods. Thus, the discrepancy between our results and values from Bartlett in terms of 2,3-DPG levels remains unexplained. Interestingly, ATP levels determined in this study are close to those reported by Bartlett.

Spectra from patients with diabetes mellitus, and even more so spectra from hyperlipidaemic patients, showed increased PDE levels and PDE/F-P-ATP ratios. Although the nature of phospholipids in blood serum has not been clarified there are hints at changes of lipid metabolism in diabetes. Hawthorne and coworkers [11, 12] have described decreased amounts of lipid inositol in brain, liver and kidney of diabetic rats, indicating reduced incorporation of phosphoesters into cell membranes.

3~p-NMR-spectra of organs contain signals of 2,3-DPG, PDE and ATP to a greater or lesser extent, and appropriate blood correction of organ spectra is thus required. For example, in heart, the creatine phosphate (CP)/ATP ratio is used as an indicator for the cardiac energy sta~e [13]. To obtain true CP/ATP ratios one has to correct for blood contamination by subtracting an appropriate amount of ATP from organ spectra (Eq. 4).

ATPco~ = ATPspectra - (2,3-DPGspectra X

ATPb]ooj2, 3-DPGblood) (4)

As data from this report show, correct use of this formula requires recognition of altered 31p-NMR resonances with disease states. Thus, ideally, one should blood correct 3~p-organ spectra after deter- mining 31p-blood resonances from the same patient. The work presented here is currently being expanded to include larger patient numbers and more disease states.

ACKNOWLEDGEMENT

This work was supported by grant No 318/3-1 from the Deutsche Forschungsgemeinschaft.

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6O

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M HORN et al.

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13. Ingwall JS (1992) in press.

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