life
ELSEVIER
Scicnrrr, Vol. 62, No. 24, pp. Wl-Wo, 1998 CopyTight0 1998 Ellevicr sciona Inc. Printed in the USA. AU *ts rcaeMd
PII SOO24-3205(98)00201-X
INTHALYMPHOCYTE
P.Delva,
FREE MAGNESIUM
C. Pastori,
OLW-3205/98 519.00 + .m
AND PLASMA TRIGLYCERIDES
M. Degan, G. Montesi and A. Lechi
Institute of Internal Medicine, University of Verona, Policlinico Borgo Roma, 37134 Verona, Italy (Received in final form March 25,1998) Summary
To evaluate the relative effect of hypertension and plasma triglycerides on intralymphocyte magnesium we measured ionized intralymphocyte magnesium (Mgi) concentration by means of a fluorimetric method based on the dye Furaptra in 4 groups of subjects: 18 normotensive normotriglyceridemic controls (NTNC) , 9 hypertriglyceridemic normotensive patients (HTN), 8 hypettriglyceridemic essential hypertensive patients (HTEH), 17 normotriglyceridemic essential hypertensive patients (NTEH). Hypercholesterolemic, diabetic patients and alcoholics were excluded from the study. Mgi was found to be statistically reduced (ANOVA test F= 10.41, P=O.OOOl) in both HTN and HTEH (M+ SD, HTN: 0.235 + 0.01, HTEH: 0.236 f 0.01 mmol/l) as compared to both NTNC and NTEH (M f SD, NTNC: 0.294 f 0.008, NTEH: 0.297+ 0.009 mmol/l). A statistically significant negative correlation was found in the population as a whole between Mgi and plasma triglycerides (n=52, R= -541, P=O.O0004). Our data suggest that hypertriglyceridemia per se and possibly the so-called plurimetabolic syndrome is characterized by low intralymphocyte free magnesium. Key Wwrk ionized magnesiuak, lymph~~
arterial hypertensioq
trigtyccrkies
Over the past decade a substantial body of experimental, epidemiological and clinical data suggests that magnesium deficiency might play a role in essential hypertension (see reference 1 for review), NIDDM (2) and in ischemic heart disease (3). A growing body of experimental and clinical data shows the importance of magnesium-dependent dyslipidaemias (4-6). Magnesium dietary deficiency in animal models exacerbates atherogenesis and lipid deposition in the arterial wall (7,8). three months’ treatment with peroral showed that after Rasmussen et al.
Correspondence to: Dr. Pietro Delva, University of Verona, Department of Internal Medicine, Policlinico Borgo Roma, 37134 Verona, Italy, tel. +39-458074414, fax: +39-45508815, E-mail:
[email protected]
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magnesium patients with ischemic heart disease showed an improvement in plasma lipid status, mainly as far as plasma triglycerides were concerned (4). Furthermore, an important feature of hyperlipemia models is the associated with magnesium deficiency in experimental animal accumulation of triglyceride-rich lipoproteins (8,9). We have previously shown that a group of hypertensive patients characterized by high plasma triglycerides demonstrated decreased levels patients with normal of lymphocyte ionized magnesium as compared to hypertensive triglycerides and to a group of normotensive normolipemic control patients (IO). From these data the relative role of hypertension and hypertriglyceridemia in intracellular magnesium depletion is not clear. To evaluate the interrelationships between hypertension, plasma triglycerides and ionized intracellular magnesium (Mg;,, we therefore studied intralymphocyte free intracellular magnesium in four groups of subjects: 18 normotriglyceridemic normotensive controls, 9 hypertriglyceridemic normotensive controls, 8 hypertriglyceridemic essential hypertensive patients and 17 normotriglyceridemic essential hypertensive patients. Methods Intralymphocyte free magnesium was measured in four groups of subjects. 18 normotensive controls with plasma triglycerides below 2 mmol/l, 9 normotensive controls with with plasma triglycerides exceeding 2 mmol/l. 8 essential hypertensive patients with plasma triglycerides exceeding 2 mmol/l and 17 essential hypertensive patients with plasma triglycerides below 2 rnmoV1. We analyzed plasma total magnesium this parameter both in the above mentioned four groups and in two groups obtained by collapsing the four groups into two groups characterized by high (n= 17) or normal plasma triglycerides (n= 35) respectively. Subjects with cholesterol exceeding 6 mmoVl were excluded from the study as were diabetic patients and alcoholics Following hospitalisation, the diagnosis of essential hypertension was formulated in all patients after a complete clinical, laboratory and instrumental examination, particular care being taken to exclude secondary forms of hypertension. The blood samples used for determining lymphocyte intracellular magnesium concentrations were taken from patients and controls in the morning after overnight fasting. All forms of drug treatment, including the use of contraceptive pills, were discontinued at least three weeks before taking the samples, but the majority of patients had never been on treatment. As diuretics are known to affect intracellular magnesium (1 l), patients on such treatment at any time were excluded from the study All subjects were on an unrestricted diet Routine Laboratory Tests Plasma total cholesterol. HDL cholesterol, triglycerides, blood glucose were measured using an autoanalyser technique (Technicon DAX 96, Miles Inc., Tarrytown, NY, USA), as was total plasma magnesium (Hitachi 911 Analyzer, Hitachi Ltd , Tokyo, Japan). HDL cholesterol was calculated using the Friedewald formula Insulin was measured by radioimmunoassay Daily 24-hour urine collections were analyzed for sodium, potassium, total magnesium and creatinine excretion. Creatinine was measured in all urine samples as an estimate of the completeness of collection. Urinary total magnesium as well as sodium and potassium were assessed by flame photometry Measurement of free intralymphocyte magnesium We used the method previously described (10). Peripheral blood lymphocytes were isolated as follows. Total blood was diluted with RPM1 1640 medium (Hepes modification, glucose-free) and layered carefully onto Histopaque 1077 and then centrifuged for 30 min at 400 ,g. The layer of lymphocytes was carefully aspirated and washed twice in RPM1 1640 for 10 min at 150 ,y in order to remove platelets. The cells thus obtained were allowed to sediment for 30 min in culture flasks. The supematant was then transferred into tubes and centrifuged, and the lymphocytes thus obtained were resuspended in the same medium. The percentage of lymphocytes thus obtained always exceeded 95% and the vitality assessed as trypan blue exclusion was always better than 97%. Lymphocytes were counted by means of a Coulter Counter (Coulter Electronics Ltd, Dunstable, Beds, U.K.). Three separate aliquots of
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Magnesium and Triglycerides
lymphocytes (6 x IO6 cells each) were suspended in RPM1 0.1% (v/v) with the cell permeant Furaptra-acetoxymethyl
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1640 with addition of BSA ester (Molecular Probes Inc.,
Eugene, OR, USA) 10 pmoU1 for 1 h at 37°C. After centrifugation, the cells were washed twice in the same medium to remove extracellular dye and resuspended in the same medium at room temperature for 45 minutes for complete deesterification of the dye For measurement of intracellular Mg*‘, the buffer utilized contained 140 mmol/l NaCl, 5 mmol/l KCI, 1.8 mmol/l CaQ, 0.8 mmol/l MgSOJ, 15 mmol/l Hepes and 5 mmol/l D-Glucose (pH 7.4 at 3 1“C). Typical fluorescence readings from furaptra-loaded cells are shown in Fig. 1. The cells, after washing off the 1640 medium, were added to the above pre-warmed medium (3 1°C) just before the fluorimetric measurements were performed. Fluorescence emission at 5 10 nm (slit width 10 nm) was measured with alternate excitation at 335 and 370 nm (slit.width 10 nm) within a thermostatically controlled cuvette holder (31°C) in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan). Autofluorecence contributed less than 1% of total fluorescence values. One cm quartz cuvettes were used for all experiments. reading After the initial from excitation at 335 and 370 nm, EDTA 5 mmol/l fluorescence emission resulting
L
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0
100
50
Time
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150
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Fig. 1 Typical experiment for intralymphocyte Mgi measurement. Fluorescence tracings of furaptra-loaded lymphocytes when excited at 335 nm (thick lines) and 370 nm (thin lines). Additions were as follows: A, (EDTA 5 mmoVl and EGTA 5 mmol/l); B, Triton X-100 (0.1 % v/v); C, MgS04 (100 mmovI).
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and EGTA 5 mmol/l were added to the cuvette and since extracellular Mg*’ and Ca*- were chelated in the medium, a rapid-step change in fluorescence at both wavelengths occurred due to dye leaking from inside the cells (see Fig. 1). The immediate (
