Vitamin B, and Hypertension“ K. DAKSHINAMURTI, C. S. PAULOSE, A N D M . VISWANATHAN Department of Biochemistry and Molecular Biology Faculty of Medicine University of Manitoba Winnipeg, Canada R 3 E 0W3 Various reports have indicated a relationship between vitamin B, status and hypertension in pregnant women and women on anovulatory steroids.’ Kleiger et a1.* have reported that the toxemic placenta is markedly deficient in pyridoxal phosphate. The lack of demonstrable therapeutic value of vitamin B, in toxemia has been ascribed to the low pyridoxine kinase activity in the toxemic placenta. Reynolds et aL3 have reported that the conversion of administered pyridoxine to pyridoxal phosphate (PLP), as reflected in the concentration of serum P L P in young preterm infants ( 1 2 9 weeks gestational age) was considerably lower than in older premature infants ( 2 3 0 weeks gestational age), thus suggesting a delay in the maturation of hepatic pyridoxine phosphate oxidase in younger premature infants. Studies in dogs4 given pyridoxine and ethanol showed decreased formation of pyridoxine to pyridoxal phosphate, and Lieber’ suggests a similar decrease in the activity of pyridoxal kinase in alcoholic humans. As suggested by Kleiger,2 a similar decrease in the conversion of pyridoxine to pyridoxal phosphate because of the decrease in the Zn2+or Mg2+ requiring pyridoxal kinase in women prone to the preeclamptic syndrome is a possibility. The EEG of an eclamptic patient who had seizures was returned to normal following administration of Mg2+and pyridoxine., Even in normal pregnancy there is a gradual decrease in serum PLP which is very significant in the third trimester. This is considerably exaggerated in the preeclamptic state. These factors confound the underlying vitamin B, status of these individuals. Apart from the preeclamptic syndrome, various clinical reports’,’ indicate a correlation between hypothyroidism and hypertension. Saito et a1.’ reported that the hypothyroid state accelerated the age-related increase in blood pressure. On thyroxine replacement blood pressure tended to normalize in the hypertensive hypothyroid patients. The mechanisms leading to hypertension in the hypothyroid condition are not known. However, in view of the relationship of hypothyroidism to vitamin B, deficiency which we have earlier shown,’ we investigated the effect of vitamin B, deficiency in the adult male rat on blood pressure regulation. Adult male Sprague-Dawley rats (6 weeks of age, 139 8 g body weight) were used in these experiments. They were randomly divided into two groups. One group was fed a pyridoxine-deficient diet and the other was fed a pyridoxine-supplemented diet and “pair-fed’’ (food intake regulated to match that of rats on the pyridoxinedeficient diet) to provide the appropriate controls. In some experiments an additional normal control, that is, rats of similar starting weight fed ad libitum a commercial rat chow for an equal length of time, was used. Systolic blood pressure was recorded OThis work was supported by a grant from the Canadian (Manitoba) Heart Foundation. 241



weekly by tail cuff plethysmography. At the end of the eight-week experimental period, direct arterial blood pressure measurements were made. Rats were anesthetized with urethane (100 mg/100 g body weight, i.p.) and the trachea was intubated in order to maintain adequate ventilation. The right carotid artery was exposed and cannulated for recording blood pressure. A 21-gauge, 2.5-cm needle with a blunt tip was fixed to the pressure transducer which in turn was connected to a Beckman Dynograph recorder R511A to record arterial pressure. Mean body weight (8) versus time (in weeks on the respective diet) curves for deficient and control rats are shown in panel B of FIGURE1. Panel A shows the mean PANEL A PYRIDOXINE SUPPLEMENTED (CONTROL)





E E v








a -a


0 0

--d m




d 8 $ P


0’ 300 PANEL B 200







FIGURE 1. (A) Mean systolic blood pressure (mmHg) versus time (weeks) for pyridoxinedeficient (n = 20) and control (n = 15) rats. (B) Mean body weight (9) versus time (weeks) for the two groups. SE bars are also given. The test rats were maintained on a pyridoxine-deficient diet from weeks 0 to 8, while control rats were maintained on a pyridoxine-supplemented diet throughout this period. At the end of the eighth week, control and pyridoxine-deficient rats were injected with pyridoxine, 10 mg/kg body weight, and blood pressure measured 24 hours later. (From Paulose er al.,” reprinted with permission of the publisher.)

systolic blood pressure (mm Hg) versus time (weeks on the respective diets) curves for these rats during the same experimental period. The blood pressure of the deficient rats had increased ( p < 0.005) by the fifth week and by the end of the experimental period, the values (143 t 6 mm Hg) were significantly higher ( p < 0.005) compared with control values (1 23 * 3 mm Hg). Twenty-four hours following treatment with pyridoxine (10 mg/kg body weight i.p.), the mean blood pressure values of the pyridoxinedeficient rats had dropped significantly to 119 * 4 mm Hg. There was no significant



TABLE I. Arterial Blood Pressure of Pyridoxine-Deficient and

Pyridoxine-Supplemented (Control) Ratsa

Animal Status Pyridoxine-supplemented (control) Pyridoxine-deficient

Diastolic Arterial Pressure (mm Hg)

Systolic Arterial Pressure (mm Hg)

77 3 105 i: 6*

111 i : 2 147 i: 5 t

aValues are means SEM of five separate determinations in each group. * p < 0.005, t p < 0.001, compared with control (by Student’s unpaired t test). (From Paulose et al.,” reprinted with permission of the publisher.)

difference between the mean blood pressure values of the control and pyridoxineinjected control rats. The specificity of the effect of pyridoxine was indicated by the fact that 4-pyridoxic acid, the biologically inactive metabolite of pyridoxine, did not have any effect on the blood pressure of control and pyridoxine-deficient rats. Direct measurement of arterial pressure by carotid artery catheterization (TABLE 1) indicates a significant increase in both systolic and diastolic blood pressure of pyridoxine-deficient rats when compared with controls. The basal heart rate was also significantly increased in pyridoxine-deficient rats as compared with appropriate controls (FIG.2). Deficient rats used in these experiments did not show outward clinical symptoms of pyridoxine deficiency. They were only moderately pyridoxine deficient as judged by the moderately lower serum PLP concentration and moderately depressed serum aminotransferase activities as compared with the control rats. FIGURE 3 shows a picture of the pyridoxine-deficient and control (pair-fed, pyridoxine-supplemented) rats. Pyridoxine deficiency is characterized by slow growth and suppressed appetite in rats. Hence it could be questioned whether the observed hypertension in the deficient rats might be a consequence of malnutrition of the deficient rats. In another experiment pyridoxine-supplemented control, ad libitum fed rats were compared with






-2 -er




Control Pyridoxine-deficient


FIGURE 2. Basal heart rate of pyridoxine-deficient and control rats at eighth week.



FIGURE 3. Appearance of pyridoxine-deficient and control rats at eighth week.

pyridoxine-supplemented rats that were “pair-weighed’’ (food intake regulated to match the weight of rats on pyridoxine-deficient diet) to examine the effect of generalized malnutrition on blood pressure. Rats subjected to generalized malnutrition had significantly lower blood pressure (87 * 4 mm Hg) compared with controls (114 & 5 mm Hg) and pyridoxine-deficient rats (149 10 mm Hg). Thus, the hypertension seen in pyridoxine-deficient rats was not a consequence of generalized malnutrition of these rats. Pyridoxine deficiency in rats could also result in a hyper-excitable state. Seizures are seen in the pyridoxine-dependent state as well as in experimental pyridoxine deficiency in young rats. Although spontaneous seizures were not observed in adult pyridoxine-deficient rats a t this stage of deficiency (however, about 30% of rats on the deficient diet for 10 weeks showed spontaneous convulsions), we investigated the effects of anticonvulsants such as phenytoin, valproic acid, and diazepam on the blood pressure of these rats. A single dose of phenytoin (6 mg/100 g body weight i.p.) decreased the systolic blood pressure in pyridoxine-deficient rats within 30 minutes from 135 4 mm H g to 105 e 3 mm Hg. The effect lasted for six hours, at the end of which blood pressure was elevated again. Valproic acid (16 mg/100 g body weight i.p.) reversed the high systolic blood pressure in the pyridoxine-deficient rats within 10 minutes from 133 3 mm Hg to 108 f 2 mm Hg. The effect lasted for only 30 minutes. In similar short-term experiments, diazepam (8 mg/100 g body weight i.p.) had no effect on the systolic hypertension of pyridoxine-deficient rats. Both valproic acid, which is supposed to act through facilitation of inhibition,” and phenytoin, which is supposed to act primarily on membranes,” produce transient pharmacological effects. In contrast, pyridoxine administration resulted in a reversal of hypertension that lasted for several days after treatment. The thyroid status of pyridoxine-deficient rats and the response to pyridoxine treatment was also examined in deficient and control (pyridoxine-supplemented) rats.



There was a significant decrease in serum thyroid stimulating hormone (TSH) ( p < 0.01), thyroxine ( p < 0.01), and triiodothyronine ( p < 0.05). There was, however, a significant increase in pituitary T S H content ( p < 0.01) in the pyridoxinedeficient rat. Treatment with pyridoxine (10 mg/kg body weight i.p.) of deficient rats reversed the hypothyroidism within 24 hours.'* W e have earlier ~ h o w n ' ~that . ' ~ the thyroid status of the pyridoxine-deficient rat is a reflection of the decrease in serotonin in the hypothalamus of these rats. There is no indication that this hypothyroid condition initiated the hypertensive condition. In view of the reported association between hypertension and increased sympathetic stimulation in patients," we investigated the possibility that the reversible hypertension seen in the deficient rat was related to general sympathetic stimulation. The concentration of norepinephrine and epinephrine in peripheral plasma can be taken as a valid reflection of sympathetic activity. However, blood samples have to be withdrawn from the conscious animal without trauma. Pyridoxine-deficient and control (pyridoxine-supplemented) rats were implanted with vascular-access ports (VAP, Model SLA, Norfolk Medical Products, Skokie, IL) with catheterization to the jugular vein.I3 The port is surgically placed subcutaneously on the back of the rat (FIG.4). The catheter is tunneled to the neck and inserted into the jugular vein. Within 24 hours rats returned to their preoperative blood pressure status (TABLE2). Deficient rats continued to be hypertensive post surgery and again, pyridoxine treatment reduced the blood pressure to normal within 24 hours. Blood samples could be collected from these animals through the VAP with minimum stress to the animals. Plasma catecholamines were extracted using alumina and assayed using an HPLC method with electrochemical detecti~n.'~.''Norepinephrine and epinephrine levels in peripheral plasma of pyridoxine-deficient rats were almost threefold higher ( p < 0.01) compared with controls (TABLE3). Treatment of deficient rats with pyridoxine (10 mg/kg body weight, i.p.) resulted in a return of systolic blood pressure to normal. Correspondingly,

FIGURE 4. Rat fitted with a vascular-access-port. (From Paulose and Dakshinamurti,I6 reprinted with permission of the publisher.)



Effect of Pyridoxine (PN) on Blood Pressure of Control and Pyridoxine-Deficient Rats


Treatment Control-before catheterization Control-after catheterization Control + PN-after catheterization Deficient-before catheterization Deficient-after catheterization Deficient + PN-after catheterization

Svstolic Blood Pressure‘(mm Hg) 119.7 i 2.3 (19) 111.8 2.1 i i 6 j 114.0 k 3.8 (6)

143.9 i 3.3f (15) 132.6 k 3.3 108.6 i 7.7

(15) (6)

‘Mean k SEM; number of animals in each group in parentheses. f p < 0.01, with respect to controls (Duncan’s multiple range test).

the plasma catecholamine levels decreased to normal levels within 24 hours of pyridoxine administration. Pyridoxine treatment of the control (pyridoxine-supplemented) rats had no significant effect on either of the indices measured. The complete reversibility of the hypertension in such a short time would preclude permanent damage to the vessel wall of the deficient animal. The lesion is probably at the level of neurotransmitter regulation. We also determined the norepinephrine turnover in the heart in both deficient and control groups by estimating the decrease in norepinephrine content” in the heart after inhibition of its synthesis with a-methyl p-tyrosine.’* There was no difference in myocardial norepinephrine content between the deficient and control groups (TABLE4). However, norepinephrine turnover in the heart was increased significantly ( p < 0.05) in the pyridoxine-deficient rats when compared to the controls, thus supporting the observation that peripheral sympathetic activity is increased in the deficient rats. The effect of anti-hypertensive drugs such as clonidine and a-methyl DOPA were tested in pyridoxine-deficient rats fitted with VAP. Basal systolic blood pressure of the deficient rat, recorded using tail cuff plethismography was stable. Following this, the anti-hypertensive drug was administered through the port twice daily at 9:OO and 17:OO hours for four days. One group of deficient rats received clonidine (10 pg/kg body weight); another received a-methyl DOPA (40 mg/kg body weight). The third group of deficient rats received the vehicle saline through the port for the same time period.

Effect of Pyridoxine on Plasma Levels of Norepinephrine and Epinephrine in Control and Pyridoxine-Deficient Adult Ratsa


Norepinephrine Epinephrine Animal Status (nmol/l) (nmol/l) Group 1: pyridoxine-supplemented(control) 3.06 r 0.28 1.89 0.28 Group 2: pyridoxine-treated (control) 3.44 0.27 1.52 0.16 9.04 i 0.21 4.39 * 0.21 Group 3: pyridoxine-deficient (experimental) Group 4: pyridoxine-treated (experimental) 3.91 0.32 2.73 + 0.24 ‘Values are means i SEM of eight to 12 separate determinations in each group; p i0.01, compared with Groups 1, 2, and 4 (by Duncan’s multiple-range test). (From Paulose et UP’; reprinted with permission of the publisher.) ~




Myocardial Norepinephrine (NE) Content and Turnover Rates in Pyridoxine-Supplemented and Pyridoxine-Deficient Adult Rats'


NE Content Animal Status (ng/g) 1. Pyridoxine-supplernented 1661.8 f 241.8 2. Pyridoxine-deficient 1955.0 f 260.8 'Values represent mean + SEM of eight separate experiments.

NE Turnover Rate

(ne/g/W 30



106.6 f 14.2'

' p < 0.05.

Blood pressure recordings were made on day 5. Both clonidine and a-methyl DOPA reduced the systolic blood pressure of deficient rats to normal levels (TABLE5). Clonidine-like drugs exert their cardiovascular depressive effects mainly through a centrally mediated sympathetic inhibition due to a stimulation of a-ad re no receptor^.'^ In view of the hypotensive effect of the az receptor agonists the possibility of decreased adrenergic output to the brain stem was examined. The kinetics of ligand binding to a2 adrenoreceptors was examined using [3H]p-aminoclonidine (PAC) binding to crude synaptasomal membrane preparations from the brain stem of pyridoxine-deficient and control rats.20Specific binding was determined by subtracting nonspecific binding from total binding. Scatchard analysis of the specific binding data was done from which maximal binding (Bmax)and the dissociation constant ( K d )were derived by linear regression analysis.2' The results (TABLE6 ) show a significant increase in the B,,, of the high- and low-affinity [3H] PAC binding to az adrenoreceptors in the brain stem of pyridoxine-deficient rats compared with controls, without any change in the binding affinity. This would indicate chronic underexposure of a2 adrenoreceptors to endogenous norepinephrine. In support of such a possibility is the significant ( p < 0.05) decrease in the MHPG levels in the brain stem of deficient rats (12.1 t 1 . 1 ng/g in deficient vs 19.4 f 1.7 in controls), indicating a low turnover of norepinephrine in the brain stem. Central GABAergic transmission has been suggested to cause hypotensive effects. Intracerebroventricular injection of GABA produces a hypotensive response" which is blocked by b i c ~ c u l l i n eAdministration .~~ of GABA or its agonists such as musimol to brain causes a reduction in blood pressure and heart rate in several specie^.^^.'^ A

Effect of Clonidine and Alpha-Methyl DOPA on Systolic Blood Pressure of Pyridoxine-Deficient Adult Rats'


1. Pyridoxine-deficient Pyridoxine-deficient


+ 4'

Clonidine (10 fig/kg) 3. Pyridoxine-deficient






Alpha-rnethyl DOPA (40 P g l W 'Values are Mean f SEM of five separate determinations. ' p < 0.01 compared with groups 2 and 3.

105 * 4



reduced sympathetic outflow has been implicated in the mediation of the cardiovascular effects of GABA.I6The central mechanism of maintaining normal blood pressure in animals is regulated by the balance between sympathetic and parasympathetic nervous The antihypertensive effect of clonidine results system tonicities in the brain from its pharmacological reactivity for central a2 adrenoreceptors in the nucleus tractus solitarri (NTS). When az adrenoreceptors in the NTS are stimulated, inhibitory neurons of the vasomotor center are activated. Sympathetic outflow, which originates from the vasomotor center and innervates the peripheral vasculature, heart, and kidney, is reduced. As a result, peripheral vascular tone, heart rate, and renin release are decreased resulting in a decrease in total peripheral resistance and cardiovascular It has been suggested that the regulation of central adrenergic receptors is not confined to adrenergic mechanisms alone but requires a serotonergic c~mponent.’~ As seen in the downregulation of p adrenergic receptors induced by anti-depressant drugs,3’modification of a, adrenergic receptors could require a degree of serotonergic input. Lesioning of the central serotonergic pathways using 5,7dihydroxytryptamine led to an increase in the B,,, of [3H]PAC binding. In the

TABLE 6. P[ 3,5,3H]-Aminoclonidinebinding to the a-Adrenoreceptors in the Brain Stem of Control and Pyridoxine-Deficient Adult Rats’

High Affinity Bm,,

Group 1. Control 2. Pyridoxine-deficient

(fmoles/mg protein) 51 89


* gb

Low Affinity

Kd (nM) 1.65 + 0.33 1.96 r 0.54

B,,, (fmoles/mg protein) 172 + 16 247 + 9b


7.48 9.17

(nM) f 1.41 * 0.47

‘Values are mean SEM of separate experiments. bp i0.01 compared with the control group.

pyridoxine-deficient rats there was a significant reduction in the serotonin content of the brain stem (3.88 f 0.14 nM/g for deficient vs. 5.39 * 0.17 for controls). The significant increase in the B,,, of ketanserin binding to the serotonin S, receptors on the crude synaptasomal membrane preparations of brain stem of the pyridoxine-deficient rat (1 57 t 8 fmoles/mg protein in deficient vs. 122 * 1 1 in controls) with no change in the binding affinity would further suggest a chronic underexposure of the brain stem to serotonergic input. This decrease in serotonergic activity of the brain stem could be responsible for the alteration in the a), adrenergic function and the resultant sympathetic stimulation. In summary, vitamin B, deficiency in the adult rat leads to hypertension resulting from general sympathetic stimulation. A central mechanism involving serotonin has been suggested to cause this sympathetic stimulation. REFERENCES 1. 2.

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Vitamin B6 and hypertension.

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