Blood pressure regulation and micronutrients.

This review attempts to delineate the underlying mechanisms leading to the development of hypertension as well as the function of vitamins and minerals in the regulation of blood pressure. Physiological processes that regulate cardiac output and systemic vascular resistance impact on the control of blood pressure. Metabolic abnormalities associated with the tetrad of hypertension, dyslipidaemia, glucose intolerance and obesity share insulin resistance, which might be organ or cell specific, as an underlying feature representing different tissue manifestation of a common cellular ionic defect. As Ca is at the centre of ionic regulation of cellular functions, vitamins involved in Ca regulation have a significant role in the control of blood pressure. The endothelium-dependent vasodilator, NO, is susceptible to oxidative damage. Hence, antioxidant vitamins and related factors regulate blood pressure through protection of NO. Robust evidence for the involvement of vitamin B6 (pyridoxine), vitamin C, vitamin D and vitamin E in the regulation of blood pressure have been reported. The well-known roles of Na, K, Ca, Mg and Cl have been explored further. The action of various vitamins on blood pressure regulation cannot always be explained on the basis of their conventionally recognised "vitamin function". The non-traditional functions of vitamins and their derivatives can be exploited as an adjunct to available pharmacological modalities in the treatment of hypertension.

Atherosclerosis risk factors: the possible role of homocysteine.

Atherosclerosis is the leading cause of death in North America. It is characterized by thickening of the coronary artery wall by the formation of plaques, resulting in reduced blood flow. Plaque rupture and the consequent thrombosis may lead to sudden blockage of arteries and causing stroke and heart attack. In the last several decades, more than 250 factors associated with the development of coronary artery disease have been identified. Recently, a relationship between atherosclerosis and elevated homocysteine level in the blood has been established. The mechanism for the production of atherosclerosis by homocysteine has been investigated. When human hepatoma cells (HepG2) were incubated with 4 mM homocysteine, enhancements in the production of cholesterol and secretion of apolipoprotein B-100 were observed. The stimulatory effect on cholesterol synthesis was mediated via the enhancement of HMG-CoA reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis. Cholesterol appears to play an important role in the regulation of apoB-100 secretion by hepatocytes. It is plausible that the increase in apoB secretion was caused by the elevated cholesterol level induced by homocysteine. The ability of homocysteine to produce a higher amount of cholesterol and promote the secretion of apoB would provide a plausible mechanism for the observed relationship between hyperhomocysteinemia and the development of atherogenesis and coronary artery disease.

Pyridoxal 5'-phosphate is an ATP-receptor antagonist in freshly isolated rat cardiomyocytes.

Although extracellular ATP is considered to exert a positive inotropic action on the myocardium through purinoceptors, very little information is available regarding interventions which may modify the actions of ATP on the heart. We report here that pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, shows antagonism towards ATP-induced positive inotropic effect in isolated perfused rat hearts, ATP-induced increase in [Ca2+] in freshly isolated adult cardiomyocytes and ATP-binding in cardiac sarcolemma; ED50 for PLP in each of these cases varied from 10-15 microM. PLP (5-50 microM) was observed to antagonize the positive inotropic effect of ATP but did not modify the action of isoproterenol in the isolated perfused heart. Preincubation of cardiomyocytes with 1-50 microM PLP prevented the ATP-induced increase in [Ca2+]i in a concentration-dependent manner but showed no effect on the KCl-induced increase in [Ca2+]i. Creatine phosphate and Na2HPO4 as well as vitamin B6-related compounds, such as pyridoxine, pyridoxal, 4-deoxypyridoxine and isonicotinic acid hydrazide showed no effect on the ATP-induced increase in [Ca2+]i in cardiomyocytes. Furthermore, different concentrations of PLP (1-50 microM) were shown to inhibit the specific ATP gamma S binding at both the high and low affinity sites in the cardiac sarcolemmal membrane; adrenoceptor and Ca2+-channel inhibitors did not affect the ATP-binding. It is concluded that PLP may antagonize the actions of ATP on the heart in a selective manner and both pyridoxal and phosphate moieties are essential for its action. Furthermore, it is suggested that PLP may serve as a valuable tool for monitoring the role of purinoceptors in cellular function.

Cardiac sarcolemmal Na(+)-Ca2+ exchange and Na(+)-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats.

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na(+)-Ca2+ exchange, Na(+)-K+ ATPase and Ca(2+)-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na(+)-K+ ATPase and Ca(2+)-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg2+ ATPase activity was not depressed throughout the experimental periods. Both Na(+)-Ca2+ exchange and ATP-dependent Ca(2+)-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na(+)-K+ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of alpha 1-subunit of Na(+)-K+ ATPase and Na(+)-Ca2+ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na(+)-K+ ATPase alpha 1-subunit mRNA was noted in diabetic heart. A significant increase in the Na(+)-Ca2+ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na(+)-K+ ATPase, Na+Ca2+ exchange and Ca(2+)-pump activities may lead to the occurrence of intracellular Ca2+ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.

Hypertension, calcium channel and pyridoxine (vitamin B6).

The moderately pyridoxine (vitamin B6)-deficient male rat was introduced by us as an animal model (B6DHT) for the study of hypertension. Hypertension in this rat is associated with increased sympathetic stimulation. Arterial segments from B6DHT rats maintained a higher resting tone. The influx of 45calcium into intracellular compartment of the vascular smooth muscle of the caudate artery of B6DHT rats was also enhanced. Administration of pyridoxine attenuated the hypertension in B6DHT rats as well as in genetic or dietary-induced moderately hypertensive conditions such as in the Zucker obese rat and sucrose or low calcium-fed rats. However, pyridoxine did not have any effect on the spontaneously hypertensive rat. All classes of calcium channel blockers were effective in lowering the systolic blood pressure of B6DHT rats. The increased in vitro influx of 45calcium into intracellular compartment of artery segments of B6DHT rats as well as the BAY K 8644-induced influx of 45calcium into artery segments from normal rats were blocked by pyridoxal phosphate as well as by dihydropyridine-sensitive calcium channel blockers (DHP). Pyridoxal phosphate (PLP) in vitro enhances the binding of calcium channel antagonists to membrane preparations from vascular tissue. PLP corrects the membrane abnormality in responsive hypertensive conditions and thus, could be an endogenous modulator of DHP-sensitive calcium channels.

Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (myoglein).

Sarcolemmal Ca2+/Mg2+ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca2+ or Mg2+ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca2+/Mg2+ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW approximately 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW approximately 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20%) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca2+/Mg2+ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca2+/Mg2+ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.

Alterations of KCl- and ATP-induced increase in [Ca2+]i in cardiomyocytes from vitamin B6 deficient rats.

Although vitamin B6 deficiency is related to coronary heart disease, no information regarding changes in myocardium due to vitamin B6 deficiency is available in the literature. In view of the critical role played by Ca2+ in cellular function, we investigated alterations in [Ca2+]i induced by KCI or ATP in vitamin B6 deficient and age-matched control rats. [Ca2+]i was measured in isolated cardiomyocytes by using the Fura-2 fluorescence technique. The KC1-induced increase in [Ca2+]i was augmented in vitamin B6 deficient cardiomyocytes, whereas the ATP-induced increase in [Ca2+]i was attenuated. The specific ATP binding to sarcolemma from hearts of vitamin B6 deficient rats was decreased. A single injection of vitamin B6 (10 mg/kg) to vitamin B6 deficient animals completely reversed the KC1- or ATP-induced changes in [Ca2+]i in cardiomyocytes as well as ATP binding with sarcolemma. These results regarding altered regulation of [Ca2+]i in cardiomyocytes and sarcolemmal ATP receptors indicate myocardial abnormalities due to vitamin B6 deficiency.

The effect of vitamin B6 on the systolic blood pressure of rats in various animal models of hypertension.

OBJECTIVE: To investigate whether a dietary supplement of vitamin B6 could attenuate the elevation of systolic blood pressure (SBP) in Zucker obese or spontaneously hypertensive rats, or rats ingesting sucrose. METHODS: Zucker obese rats (fa/fa), Sprague-Dawley rats with sucrose-induced elevation of SBP, spontaneously hypertensive rats (SHRs) and their corresponding controls were tested for the effects of vitamin B ingestion in different ways: (1) vitamin B6 was included as a supplement (five times the normal intake) from the start of the experiment until the development of hypertension; (2) vitamin B6 supplement was removed from the diet of Zucker obese and Zucker lean control groups after 16 weeks on the dietary treatments; and (3) a diet deficient in vitamin B6 was instituted in SHRs and control Wistar-Kyoto (WKY) rats. The SBP of rats in all groups was monitored in the conscious animal by tail-cuff plethysmography. The effects of the various treatments on the uptake of calcium by caudal artery segments were examined. RESULTS: Male Zucker obese rats (fa/fa) of age 6 weeks fed a commercial rat chow developed hypertension in 3-4 weeks, whereas their lean controls (Fa/Fa) did not. The inclusion of a vitamin B6 supplement (five times the normal intake) resulted in a complete attenuation of the hypertension in the obese strain. Removal of the vitamin B6 supplement from the diet of these obese rats resulted in the return of hypertension within 2 weeks. Similar changes in SBP were also observed in the Zucker lean controls treated with vitamin B6. The ingestion of sucrose by male Sprague-Dawley rats resulted in modest elevation of SBP that was attenuated by the inclusion of the vitamin B6 supplement in their diet. In contrast, there was no response to the inclusion or removal of dietary vitamin B6 supplement in the SHRs. However, the WKY control rats responded to both these conditions in a similar manner to that seen in the Sprague-Dawley strain. Increased peripheral resistance resulting from increased permeability of vascular smooth muscle plasma membrane to Ca2+ is thought to be one of the mechanisms of hypertension. Changes in SBP correlated with changes in the uptake of calcium by caudal artery segments in all the groups studied. The Zucker obese and sucrose-induced hypertensive rats have abnormalities in carbohydrate metabolism. The vitamin B6 supplement decreased the random or fasting blood glucose levels in the Zucker obese and sucrose-fed rats respectively. CONCLUSION: This is the first observation that animal models of hypertension can be classified on the basis of their response to a vitamin B6 supplement. On this basis, the etiology of hypertension in SHRs is quite distinct from that in Zucker obese rats and in rats ingesting sucrose.

Biotin influences palatal development of mouse embryos in organ culture.

Maternal biotin deficiency is strongly teratogenic in CD-1 mice. The most common malformations are craniofacial and limb defects such as cleft palate, micrognathia and micromelia. The effect of biotin deficiency on palatal development in mouse embryos on d 12 of gestation was studied by culturing mouse embryonic palates in serum-free medium using a suspension culture system. In control embryos palatal processes developed to the fused stage after 72 h in culture. The fusion of palatal processes was further increased by the addition of biotin (10(-8) mol/L) to the medium. The addition of organic acids such as propionic, beta-methyl crotonic or beta-hydroxy isovaleric acids as well as avidin to the medium did not affect the stage of palatal formation. Cycloheximide completely blocked the fusion of palatal shelves. In embryos from biotin-deficient mice, the incidence of fusion between the palatal shelves was < 7% and increased to > 30% when biotin (10(-8)-10(-6) mol/L) was added to the medium. The addition of fatty acids to the organ culture medium did not have any effect on the fusion of palatal processes. The incorporation of 35S-methionine into protein from biotin-deficient embryo explants was 88% of that in controls. The results indicate that biotin deficiency may interfere directly with synthesis of specific proteins and the formation of palatal processes.

The relationship between low-calcium-induced increase in systolic blood pressure and vitamin B6.

OBJECTIVE: To investigate the relationship between the increase in systolic blood pressure caused by a low-calcium (0.1%) diet and the vitamin B6 status in the rat. METHODS: Male Sprague-Dawley rats fed a vitamin B6-deficient diet did not show any change in systolic blood pressure (SBP) for the first 4 weeks (prehypertensive phase), but from week 5 there was an increase in SBP lasting until week 10 (hypertensive phase). SBP declined to below normal levels in most rats from week 12 on the vitamin B6-deficient diet (posthypertensive phase). The effect of altering the level of calcium in the diet at different phases of vitamin B6 deficiency was studied. In another experiment the effect on the change in blood pressure induced by the low-calcium diet of increasing the dietary vitamin B6 level to 2.5-, 5- or 10-fold the normal intake was studied. RESULTS: Lowering dietary calcium caused a significant increase in SBP in rats on the vitamin B6-sufficient diet. This occurred during weeks 3 and 4 on the low-calcium diet. Low levels of calcium on the diet potentiated the hypertension induced by the vitamin B6-deficient diet when both deficiencies were present from the beginning of the experiment. Feeding a low-calcium diet during the hypertensive or posthypertensive phase failed to raise the SBP in these rats. Normalizing the vitamin B6 status of posthypertensive vitamin B6-deficient rats restored the ability of low dietary calcium to increase SBP in these rats. Increasing dietary levels of vitamin B6 by itself reduced SBP in normal rats, and attenuated the increase in SBP induced by the low-calcium diet. CONCLUSIONS: Dietary vitamin B6 deficiency and low calcium in the diet seem to share the mechanisms increasing SBP.

Effects of serotonergic agents on plasma prolactin levels in pyridoxine-deficient adult male rats.

Plasma concentration of prolactin was significantly reduced in pyridoxine-deficient as compared to control (pyridoxine-supplemented) adult male rats. Administration of pyridoxine to deficient rats resulted in a significant increase in plasma prolactin. The reduction in plasma prolactin in pyridoxine-deficient rats corresponded with the significantly reduced hypothalamic contents of pyridoxal phosphate and serotonin in pyridoxine-deficient rats. Plasma prolactin concentrations were also measured in response to serotonergic agents in both groups of rats. The administration of the 5HT1A agonist (8-hydroxy 2-n-dipropylamino tetralin) resulted in a significant increase in plasma prolactin and that of the specific 5HT1A antagonist spiroxatrine had the opposite effect. The results suggest that the hypothalamic serotonergic regulation of prolactin release is impaired in pyridoxine deficiency.

Transcriptional regulation of liver phosphoenolpyruvate carboxykinase by biotin in diabetic rats.

Rat liver phosphoenolpyruvate carboxykinase (PEPCK) activity was followed over a time period of 5 h following administration of biotin to streptozotocin-induced diabetic rats. In parallel with the decrease in enzyme activity liver PEPCK mRNA decreased by 85% at 3 h after injection of biotin to diabetic rats. There was no significant change in the accumulation of kidney PEPCK mRNA. Parallel studies with insulin indicated that biotin had a regulatory effect similar to that of insulin on liver PEPCK mRNA. The administration of biotin did not change the insulin status of the diabetic rat indicating that biotin did not act via insulin. The transcriptional activity of the hepatic PEPCK gene, as measured by nuclear run-on assay, was decreased by 57% within 30 min of biotin administration. The results suggest that biotin regulates hepatic, but not renal, PEPCK mRNA concentration at the transcriptional level in diabetic rats.

Picrotoxin and pentylene tetrazole induced seizure activity in pyridoxine-deficient rats.

Computerized electroencephalography and thalamic ventro-posterior lateral (VPL) unit activities were recorded from pyridoxine-deficient and pair-fed pyridoxine-supplemented adult male rats. Pyridoxine-deficient animals exhibited slow electroencephalograms (EEG) represented by the dominance of delta activity and reduced seizure thresholds to local (VPL) application of either picrotoxin or pentylene tetrazole. Frequency and amplitude of thalamic VPL unit activity were significantly reduced in pyridoxine-deficient rats as compared to pyridoxine-supplemented controls. Pyridoxine-deficient rats exhibited irregular unit activity with frequent bursts and electrosilent periods in response to local (VPL) picrotoxin or pentylene tetrazole microinjections. They also exhibited severe seizure discharge activity of prolonged duration at any given dose of either picrotoxin or pentylene tetrazole. This was represented by significantly increased burst frequency, burst duration and reduced seizure latencies. Unit activity was transformed into burst discharge activity with intermittent electrosilent zones during picrotoxin or pentylene tetrazole epileptogenesis. Cerebral gamma aminobutyric acid (GABA) level was reduced and glutamate concentration increased in pyridoxine-deficient rats when compared with pyridoxine-supplemented controls. Local (VPL) microinjection of GABA or pyridoxine induced neuronal recovery in both convulsant-treated normal and pyridoxine-deficient rats. Neuronal recovery was however delayed in pyridoxine-deficient rats. Neuronal recovery was associated with a significant increase in EEG background frequency and reduction in delta frequencies in the EEG records of both normal and pyridoxine-deficient rats. Reduced seizure threshold and delayed neuronal recovery are related to the significantly reduced brain regional GABA and elevated glutamate levels in pyridoxine-deficient rats.

Calcium channels in vitamin B6 deficiency-induced hypertension.

OBJECTIVE: To compare the acute hypotensive effects of various calcium channel modulators in vitamin B6-deficient hypertensive (B6DHT) rats. METHODS: Adult male Sprague-Dawley rats were fed a vitamin B6-deficient diet for 7-10 weeks, during which systolic blood pressure (SBP) was measured in the B6DHT and control rats, using tail-cuff plethysmography. The effects of the calcium antagonists nifedipine, verapamil, diltiazem and (-)-202-791 on SBP were determined in conscious B6DHT rats. The effect of the calcium agonist BAY K 8644 on the SBP of rats fed various levels of pyridoxine was also determined. RESULTS: All of the calcium antagonists used were effective in lowering the SBP of the B6DHT rats with the rank order of potency: nifedipine > (-)-202-791 > (+/-)-verapamil > diltiazem. BAY K 8644 elevated the SBP of older rats fed a normal commercially available diet, but had no effect when the vitamin B6 content of their diet was increased or removed for a short period. CONCLUSION: Calcium channel function appears to be related to vitamin B6 status in the rat.

Hippocampal changes in developing postnatal mice following intrauterine exposure to domoic acid.

Domoic acid (0.6 mg/kg) was injected intravenously through the caudal vein in pregnant female mice on the 13th day of gestation and EEG was monitored in the developing progeny during postnatal days 10-30. No clinical seizure activity was observed during this period. However, these mice demonstrated generalized electrocortical inhibition associated with diffuse spike and wave activity in their basal EEG records. Intrauterine domoic acid-exposed (IUD) mice had significantly reduced seizure thresholds to an additional dose of domoic acid, given postnatally. At the light microscopic level, hippocampus of IUD mice exhibited age related developmental neurotoxicity. No cellular damage was observed on postnatal day 1. On day 14, severe neuronal damage was observed in the hippocampal CA3 and dentate gyrus regions. On day 30, in addition to CA3 and dentate gyrus, CA4 was also involved. Brain regional GABA levels were significantly reduced and glutamate levels increased in IUD mice. Kainate receptor binding to hippocampal synaptosomal membranes from IUD mice at 30 d of age was significantly increased. There was also an enhanced 45Ca influx into cortical and hippocampal slices of these mice. These findings suggest that intrauterine exposure to domoic acid can induce hippocampal excitotoxicity by increasing the neuronal calcium influx through kainate receptor activation. Histological changes suggest progressive hippocampal damage in IUD mice, but without overt clinical seizures.

Regulation of calcium influx into vascular smooth muscle by vitamin B6.

In the vitamin B6 deficiency-induced hypertensive (B6DHT) rat there is an increased influx of calcium into the vascular smooth muscle. Vitamers which possess vitamin B6 activity blocked the in vitro calcium influx into the caudal artery of B6DHT rats and the BAY K 8644-induced influx into artery segments from vitamin B6-supplemented (control) rats. BAY K 8644 did not increase further the calcium influx into caudal artery segments from B6DHT rats. However, when this influx was partly decreased by Nifedipine or by pyridoxal phosphate, BAY K 8644 antagonized their effects indicating that calcium influx mediated by the dihydropyridine-sensitive calcium channel is a site of the calcium influx defect in the B6DHT rat.

Hypotensive action of 5-HT receptor agonists in the vitamin B6-deficient hypertensive rat.

Feeding a vitamin B6-deficient diet to rats causes a moderate hypertension. The blood pressure responses to 5-HT1A receptor agonists were studied in conscious vitamin B6-deficient hypertensive rats. They were all effective in lowering blood pressure with the following rank order of potency: 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) > flesinoxan > 5-methylurapidil > urapidil. The putative 5-HT1A receptor antagonist spiroxatrine by itself, did not have any effect on the blood pressure at the doses used (0.01-1 mumol/kg). However, dose dependently, it antagonized the hypotensive effect of flesinoxan and urapidil. The alpha 1-adrenoreceptor antagonist prazosin, on prior treatment, did not change the hypotensive effect of either flesinoxan or urapidil. The alpha 2-adrenoreceptor agonist clonidine dose dependently (0.01-0.1 mumol/kg) reduced blood pressure. This effect of clonidine was unaffected by spiroxatrine, but was dose dependently antagonized by the alpha 2-adrenoreceptor antagonist yohimbine. Binding studies with [3H]8-OH-DPAT indicated that the affinity and Bmax of 5-HT1A receptors was increased in vitamin B6-deficient hypertensive rats. The results suggest that decreased synthesis of 5-HT in brain regions and the consequent alterations in 5-HT receptors in the vitamin B6-deficient rats may be the underlying cause of the hypertension seen in these animals.

Suppression of domoic acid induced seizures by 8-(OH)-DPAT.

Microinjections of the neuroexcitotoxin, domoic acid (DOM), in the ipsilateral rat hippocampal CA-3 region, induced generalized electrical seizure discharge activity, characterized by spikes and waves, followed by intermittent burst discharges. Computerized EEG analysis exhibited relative dominance of delta and theta and reductions in alpha and beta activities during domoic acid epileptogenesis. Seizure discharge activity was attenuated by the microinjection of the 5-HT1A agonist, 8-hydroxy-2-(di-N-propylamino)tetralin(8-(OH)-DPAT) and augmented by the specific 5-HT1A antagonist, spiroxatrine in the contralateral hippocampal CA-3 region. Neuronal recovery following 8-(OH)-DPAT was associated with significant reductions in the relative dominance of delta and theta and increases in the alpha and beta activities. The results suggest that activation of serotonergic 5-HT1A receptor in the hippocampus has a neuroprotective action.

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.

Seizure activity in pyridoxine-deficient adult rats.

This investigation tested the hypothesis that the degree of pyridoxine depletion rather than the status of neuronal maturity determines seizure proneness in the pyridoxine-deficient rat. Dietary pyridoxine deficiency was induced in neuronally mature rats. Seizure activity was monitored using computerized EEG analysis. Dietary pyridoxine deficiency of 10 weeks' duration induced in neuronally mature rats led to spontaneous convulsive seizure activity. Even moderately pyridoxine-deficient adult rats (on the deficient diet for less than 8 weeks) exhibited seizure-like diffuse spike and wave activity and electrocortical inhibition. Picrotoxin-, pentylenetetrazol-, or domoic acid-induced seizure thresholds were significantly reduced in pyridoxine-deficient rats when compared with normal controls. Pyridoxine-deficient rats exhibited increased dominance of delta and theta activities and increased hemispherical asymmetries in response to convulsant treatment.