There is a well known excess of hypertension. Serum Ionized Magnesium. Relation to Blood Pressure and Racial Factors

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1 AJH 1997;10: Serum Ionized Magnesium Relation to Blood Pressure and Racial Factors Lawrence M. Resnick, Orit Bardicef, Bella T. Altura, Michael H. Alderman, and Burton M. Altura To study potential ionic factors predisposing to vascular disease in hypertension, particularly among black subjects, we used a recently developed combined magnesium and calcium specific, ion selective electrode apparatus to measure extracellular ionized calcium (Ca-ion), ionized magnesium (Mg-ion), and Ca-ion/Mg-ion ratios in the serum of fasting, nonmedicated white and black normotensive (n 61) and hypertensive (n 23) subjects, studied consecutively in a tertiary referral center. Both race and blood pressure status had independent effects on the distribution of Mg-ion values. Although Mg-ion levels for the group as a whole were lower in hypertensive versus in normotensive subjects ( v mmol/l; P <.01), this was only true of white subjects ( v mmol/l; P.0095). The lack of a significant difference in Mg-ion levels between black hypertensive versus normotensive subjects ( v mmol/l, P NS) was attributable to the significantly lower Mg-ion levels present in normotensive blacks compared to those in normotensive white subjects ( v mmol/l, P.0001). Resultant Ca-ion/ Mg-ion ratios were elevated in all black subjects and in white hypertensive subjects. These data support the presence among hypertensives and among black subjects (independently of blood pressure) of a consistent depletion of circulating magnesium and of an imbalance of calcium and magnesium that may potentiate vascular disease among these subjects. Am J Hypertens 1997;10: American Journal of Hypertension, Ltd. KEY WORDS: Magnesium, blood pressure, race, calcium, blacks. There is a well known excess of hypertension and its sequelae in the black population. This predisposition extends not only to the prevalence and severity of hypertension, 1 but to the organ system complications associated with it, including left ventricular hypertrophy, 2 hypertensive nephropathy, 3 stroke, 4 and overall cardiovascular mortality. 5 Despite these consistent epidemiologic data, the biologic basis underlying this excess morbidity and mortality in black populations remains undefined. Our group has attempted to understand hypertension and its cardiovascular complications by studying mineral ion metabolism and its hormonal and intra- Received January 16, Accepted July 15, From the Cardiovascular Center, New York Hospital-Cornell Medical Center, New York, New York (LMR); Department of Physiology, State University of New York, Brooklyn, New York (BTA, BMA); Department of Epidemiology, Albert Einstein College of Medicine, Bronx, New York (MHA), and Division of Endocrinology and Hypertension, Wayne State University Medical School, Detroit, Michigan (LMR, OB). Address correspondence and reprint requests to Lawrence M. Resnick, MD, Division of Endocrinology and Hypertension, Wayne State University Medical School, University Health Center 4H, 4201 St. Antoine, Detroit, MI by the American Journal of Hypertension, Ltd /97/$17.00 Published by Elsevier Science, Inc. PII S (97)

2 AJH DECEMBER 1997 VOL. 10, NO. 12, PART 1 SERUM IONIZED MAGNESIUM IN HYPERTENSION 1421 cellular consequences. 6 Specifically, we have demonstrated the presence of excess intracellular free calcium levels and suppressed free magnesium levels in hypertension that were closely linked to the height of the blood pressure, to the degree of cardiac hypertrophy, and to the hyperinsulinemia and insulin resistance characteristic of hypertension. 7 Indeed, we have suggested an ionic hypothesis in which hypertension, as well as its metabolic and cardiovascular consequences, are all different clinical manifestations of a common defect in cell ion handling. 8 We wondered to what extent these concepts might also provide insights into the greater prevalence and pathologic consequences of hypertensive vasculopathies among black subjects. Indeed, magnesium deficiency, as well as calcium excess have been implicated in the pathogenesis of hypertension and atherosclerosis. 9,10 Therefore, we studied the distribution of extracellular ionized calcium and magnesium in fasting black and white subjects, using a newly available ionspecific electrode apparatus. 11 Our preliminary results demonstrate that both elevated blood pressure and racial factors are associated with an altered distribution of serum ionized magnesium, but not ionized calcium values. Thus, hypertension is associated with lower Mg-ion values, and black individuals, independently of their blood pressure status, exhibit lower serum ionized magnesium levels compared with white control subjects. We suggest that the resulting increased calcium:magnesium ratio among these groups may contribute to the incidence and severity of vascular disease in black or hypertensive populations. METHODS Nonmedicated normotensive and essential hypertensive black and white subjects free of other known diseases were studied after an overnight fast at the Cardiovascular Center of the New York Hospital Cornell Medical Center. Normotensive subjects were recruited from among a working population designed to reflect the demographic characteristics of the general population in New York City, as part of a prospective study on the incidence of cardiovascular disease in a general population. Essential hypertensive subjects were diagnosed on the basis of elevated blood pressure readings found repeatedly to be 150/95 mm Hg, in the absence of physical diagnostic or laboratory evidence of secondary hypertension. Blood samples for analysis (see below) were obtained as part of ongoing protocol studies approved by the Scientific Advisory and Human Rights committees. Blood was drawn in the seated position in the Cardiovascular Center of the New York Hospital Cornell Medical Center. Blood samples were analyzed for serum total and ionized magnesium (Mg-ion) and for serum ionized calcium (Ca-ion). Total magnesium levels were measured by standard automated techniques. Blood for serum ionized magnesium (Mg-ion) was drawn into air evacuated glass tubes containing an inert cell separating matrix. After clotting and centrifugation the tubes were inverted and serum was drawn off into a syringe anaerobically, the latter being capped and sent for analysis. A magnesium ion selective electrode (ISE) with a neutral carrier based membrane was used to measure serum ionized magnesium. As previously described, 11 this new ISE for free magnesium gives equivalent values of Mg-ion in samples of blood, serum, or plasma. The same selective ion apparatus, equipped with a calcium-specific electrode, was also used to concurrently measure serum ionized calcium values. All data were analyzed by two-way analysis of variance (ANOVA), with statistical significance assessed by post hoc t tests for all means (Bonferroni-Dunn) to test the effects of both blood pressure status (hypertensive versus normotensive subjects) and race (black versus white subjects) (Super Anova, Abacus Concepts, Berkeley, CA). All data are reported as the means SEM. RESULTS The age, sex distribution, weight, and renal function of the hypertensive subjects did not differ significantly between black versus white subjects, or between normotensive versus hypertensive individuals. Both systolic and diastolic pressure were elevated in hypertensive compared with normotensive controls, but did not differ between black versus white subjects in either blood pressure group. For the group as a whole, independent of race, Mg-ion values were significantly lower in hypertensive (n 23, HiBP), compared with normotensive (n 61, NlBP) subjects ( v mmol/l, P.01). When analyzed further, however, it was observed that this difference was only significant among white individuals (NlBP: v HiBP: mmol/l, P.0095), but not among black subjects, in whom Mg-ion levels among normotensive and hypertensive individuals did not significantly differ (NlBP: v mmol/l, P.1609) (Table 1). Indeed, the most significantly different results were observed in normotensives, among whom black subjects exhibited significantly lower Mg-ion values than did whites ( v mmol/l, P.0001 (Figure 1A). There were no significant differences in male versus female subjects, between either different blood pressure or racial groups. These results were also observed when serum total magnesium values were analyzed; lower values were found among black versus white normotensives ( v

3 1422 RESNICK ET AL AJH DECEMBER 1997 VOL. 10, NO. 12, PART 1 TABLE 1. SERUM LEVELS OF TOTAL MG (MG-TOT), IONIZED MG (MG-ION), AND IONIZED CALCIUM/IONIZED MG RATIOS IN NORMOTENSIVE (NlBP) AND HYPERTENSIVE (HiBP) SUBJECTS GROUPED ACCORDING TO RACE Group Mg-tot (mmol/l) Mg-ion (mmol/l) Ca-ion/Mg-ion White NlBP HiBP Black NlBP * * * HiBP *P.0001 v white subjects; P.01 v NlBP mmol/l, P.0001), but not hypertensives ( v , NS). Fasting serum ionized calcium levels, obtained concurrently, did not differ among any of the groups, being the same for normotensives as a whole versus hypertensives ( v mmol/l, P.713), for white normotensives versus hypertensives ( v mmol/l, P.5217), and for black versus white normotensives ( v mmol/l, P.7632). Ratios of Ca-ion to Mg-ion were also calculated as an index of the net divalent ion contribution to vascular tone. 12 A greater Ca-ion/Mg-ion ratio was found in hypertension compared with normotensives for all subjects (HiBP: v NlBP: , P.004), and among hypertensive v normotensive whites (HiBP: v NlBP: , P.0025), but not blacks (HiBP: v , P.1184). This was again because normotensive black subjects already had an elevated Ca-ion/Mg-ion ratio compared with similarly normotensive whites (blacks: v , P.0001) (Figure 1B). DISCUSSION Hypertension, as well as its clinical-pathological consequences, are more prevalent in black populations, and are associated with an earlier onset, increased severity, and greater target organ damage. 1 Mild to moderate hypertension is 1.5 to 2 times more prevalent in blacks than in whites, with severe hypertension being 5 to 7 times more prevalent. 1 The annual rate of referral for end-stage renal disease in one study was 4.2 times greater for blacks than for whites, and in hypertensives, was 17.7 times higher in blacks than in whites. 3 Left ventricular hypertrophy, by electrocardiographic criteria, exhibits a 2- to 4-fold greater prevalence in hypertensive blacks compared with their white counterparts. 2 Stroke is 2 to 3 times more likely in black versus white populations, even without concurrent hypertension. 4 Despite these consistent epidemiologic data, the biologic basis underlying this excess morbidity and mortality in black populations has remained undefined. Our approach to this problem has emphasized the role of mineral ions in general, and of magnesium in particular, in the pathophysiology of hypertension, insulin resistance, and diabetes mellitus. 6,7,9 We have focused on magnesium as well as calcium because magnesium is the major intracellular divalent cation, regulating enzyme function, modulating cellular responsiveness to physiological stimuli such as calcium, and thus steady state organ activities including car- FIGURE 1. Serum ionized magnesium values (Mg-ion, left, A) and the ratio of serum ionized calcium to serum ionized magnesium (Ca-ion/ Mg-ion, right, B) in normotensive white versus black subjects.

4 AJH DECEMBER 1997 VOL. 10, NO. 12, PART 1 SERUM IONIZED MAGNESIUM IN HYPERTENSION 1423 diac output, vasoconstrictor tone, pancreatic insulin secretion, and peripheral insulin sensitivity. 9,13 In normal populations, lower serum magnesium levels with age are associated with higher blood pressures. 14 Pathologically, both essential hypertension and non insulin-dependent diabetes mellitus (NIDDM) are associated with significantly suppressed intracellular free magnesium levels, 7 which, in turn, are inversely related to the height of the pressure and the degree of insulin resistance the lower the magnesium, the higher the pressure and the greater the hyperinsulinemic response to oral glucose loading. Consistent with this, the ability of oral and parenteral magnesium administration to lower blood pressure and to ameliorate diabetes have been long known and have recently been rediscovered. 15 Furthermore, experimentally induced magnesium deficiency can cause vasoconstriction, increase platelet aggregation, produce insulin resistance, and accelerate atherosclerosis. 9,10,16 Altogether these observations led us to reason that chronically lower steady state levels of magnesium, or an imbalance between circulating calcium and magnesium, 9,11 might predispose to and potentiate hypertensive, diabetic, or atherosclerotic vascular disease. As such, we hypothesized that these alterations would most likely be found in patient groups predisposed to or suffering from these diseases. Our results suggest that this may indeed be the case. Technically, the lack of a routinely available, accurate, and physiological measurement of magnesium has long hampered progress in this area. The measurement of free cytosolic magnesium has only recently been possible with the advent of nuclear magnetic resonance (NMR) spectroscopic techniques, 17 and fluorescent probes. In the extracellular space, although serum ionized calcium measurements have long been available, we have only recently described a new, ion specific electrode apparatus for measuring serum ionized magnesium in healthy subjects. 11 Using this technique, we have demonstrated significant suppression of circulating ionized magnesium in mild, fasting diabetic subjects, even when no such differences in serum total magnesium were apparent. These extracellular magnesium levels were, in turn, closely related to concurrently measured intracellular free magnesium values. 18 In this study, we used ion specific electrode techniques to measure both serum ionized calcium and magnesium in normotensive and in hypertensive subjects. We observed: 1) lower circulating levels of serum ionized magnesium in white individuals with essential hypertension, compared with normotensive white control subjects; 2) racial differences in serum ionized magnesium levels among normotensive subjects in otherwise healthy black adults having lower serum ionized magnesium levels compared with those of normotensive white control subjects; and 3) a higher ratio of serum ionized calcium to serum ionized magnesium in hypertensive versus normotensive white subjects and in black versus white normotensives. These data further support the association of magnesium depletion with hypertension and suggest that racial differences in magnesium status may also help to explain the predisposition of black populations to vascular diseases and their complications. This latter notion is further supported by the finding of higher Ca-ion/Mg-ion ratios in the same patient groups having lower absolute Mg-ion levels, as this ratio has been directly related to the potential for arterial calcification in experimental models of atherosclerosis. 10,11 A few caveats need to be considered in interpreting these preliminary data. First, because lower magnesium levels were found in black subjects in the absence of overt hypertensive disease, magnesium depletion of this modest degree cannot per se account for hypertension, with other as yet undefined concomitant factors presumably needed for the emergence of elevated blood pressure. This is also consistent with suppressed levels of intracellular free magnesium found in other nonhypertensive conditions such as non insulin-dependent diabetes mellitus. 6,8,18 We have not assessed intracellular free magnesium values in a sufficient number of normotensive and hypertensive black subjects to be able to determine whether the equally low serum ionized magnesium values in normotensive and hypertensive black subjects are also true for the intracellular ionic species. Thus, lower magnesium levels, whether intracellular or extracellular, may be a necessary but not sufficient condition for the onset of hypertension. Second, the use of serum ionized magnesium measurements as an accurate reflection of tissue magnesium levels needs to be better documented. The close correspondence between intracellular free magnesium levels, as assessed by NMR spectroscopy in peripheral red blood cells, and the concurrently measured serum ionized magnesium level 18 needs to be extended to the evaluation of intracellular free magnesium levels in organ systems that are critical targets for hypertensive and diabetic vascular disease, such as brain, heart, and kidney. Preliminary reports in essential hypertensive subjects suggest an intracellular free magnesium deficit in situ in brain and skeletal muscle, similar to previous reports in peripheral blood cells. 19 Third, these measurements, both serum ionized magnesium and intracellular free magnesium, as well as serum ionized calcium, all need to be made in the fasting state, as was done here. This is because intracellular free magnesium and calcium levels change following oral glucose ingestion and in vitro glucose and insulin incubation, 20 which appears to be equally and inversely true for serum magnesium and calcium in the extracellular

5 1424 RESNICK ET AL AJH DECEMBER 1997 VOL. 10, NO. 12, PART 1 compartment. 21 Failure to evaluate subjects in the fasting state may thus obscure differences between hypertensive and normotensive subjects. Fourth, much larger numbers of subjects will need to be evaluated to confirm and extend these findings. Lastly, the pathophysiologic mechanism(s) underlying these data is unknown, and measurements of dietary mineral intake, circulating mineral regulating hormones, other markers of overall mineral balance, and other factors not measured in this preliminary study, whether dietary environmental or genetic physiologic, need to be considered in future studies. REFERENCES 1. Saunders E: Hypertension in blacks. Primary Care 1991;18: Beaglehole R, Tyroler HA, Cassel JC, et al: An epidemiological study of left ventricular hypertrophy in the biracial population of Evans County, Georgia. J Chron Dis 1975;28: Rostand SJ, Kirk KA, Rutsky EA, Pate BA: Racial differences in the incidence of treatment for end-stage renal disease. N Engl J Med 1982;306: Modan B, Wagener DK: Some epidemiological aspects of stroke: mortality/morbidity trends, age, race, socioeconomic status. Stroke 1992;23: Francis CK: Making a difference. Managing hypertension in minority patients. Am J Med 1990;88(suppl 3B):1S 2S. 6. Resnick LM: Cellular ions in hypertension, insulin resistance, obesity and diabetes: a unifying theme. J Am Soc Nephrol 1992;3:S78 S Resnick LM, Gupta RK, Laragh JH: Intracellular free magnesium in erythrocytes of essential hypertension: relation to blood pressure and serum divalent cations. Proc Natl Acad Sci USA 1984;81: Resnick LM: Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders: mechanism of syndrome X. Am J Hypertens 1993;6:123s 134s. 9. Altura BM, Altura BT: Magnesium ions and contraction of vascular smooth muscle: relationship to some vascular diseases. Fed Proc 1981;40: Altura BT, Brust M, Bloom S, et al: Magnesium dietary intake modulates blood lipid levels and atherogenesis. Proc Natl Acad Sci USA 1990;87: Altura BT, Shirey TL, Young CC, et al: A new method for the rapid determination of ionized Mg 2 in whole blood, serum and plasma. Meth Find Exper Clin Pharmacol 1992;14: Nakayama K, Fleckenstein A, Byon YK, Fleckenstein- Grun G: Fundamental physiology of coronary smooth musculature from extramural stem arteries of pigs and rabbits (electric excitability, tension development, influence of Ca, Mg, H and K ions). Eur J Cardiol 1978; 8: Gueux E, Rayssiguier Y: The effect of Mg deficiency on glucose stimulated insulin secretion in rats. Horm Metab Res 1983;15: Peterson B, Schroll M, Christiansen C, Tranavol I: Serum and erythrocyte magnesium in normal elderly Danish people. Acta Med Scand 1977;201: Paolisso G, Sgambato S, Pizza G, et al: Improved insulin response and action by chronic magnesium administration in aged NIDDM subjects. Diabetes Care 1989; 12: Nadler JL, Malayan S, Luong H, et al: Intracellular free magnesium deficiency plays a key role in increased platelet reactivity in type 2 diabetes mellitus. Diabetes Care 1992;15: Gupta RK, Benovic JL, Rose JB: The determination of the free magnesium level in the human red blood cell by 31 P-NMR. J Biol Chem 1978;253: Resnick LM, Altura BT, Gupta RK, et al: Intracellular and extracellular magnesium depletion in non insulin dependent diabetes mellitus. Diabetologia 1993;36: Resnick LM, Bardicef O, Barbagallo M, et al: 31 P-NMR spectroscopic studies of oral glucose loading and in situ skeletal muscle ion content in essential hypertension (abst). Hypertension 1995;26: Resnick LM, Gupta RK, Laragh JH: Possible effects of diet and other factors on 31 P-nuclear magnetic resonance measurements of intracellular magnesium in hypertension. Clin Sci 1989;73: Rosenboom AL: Serum calcium and magnesium decline during oral glucose tolerance testing in children with preclinical diabetes mellitus, less than in normals. Metabolism 1977;26: