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Biomedical subjects

P Smits

Publications and source records attributed to P Smits.

At least 73 records · Page 4Linked to original sources
PubMed2000

Comparison of two indices for forearm noradrenaline release in humans.

Although there is as yet no method which measures directly the neuronal release of noradrenaline in humans in vivo, the isotope dilution technique with [(3)H]noradrenaline has been applied to estimate forearm neuronal noradrenaline release into plasma. Two different equations have been developed for this purpose: one to estimate the spillover of noradrenaline into the venous effluent, and a modified formula (often referred to as the appearance rate) which may reflect more closely changes in the neuronal release of noradrenaline into the synaptic cleft, particularly during interventions that alter forearm blood flow. The present study was performed to compare the effects of two interventions known to exert contrasting actions on neuronal forearm noradrenaline release and forearm blood flow. Intra-arterial infusion of sodium nitroprusside at doses without systemic effect increases forearm blood flow, but not neuronal noradrenaline release. In contrast, lower-body negative pressure at -25 mm Hg causes forearm vasoconstriction by stimulating neuronal noradrenaline release. During sodium nitroprusside infusion, forearm noradrenaline spillover increased from 1.1+/-0.3 to 2.2+/-1.0 pmol x min(-1) x 100 ml(-1) (P<0.05), whereas the forearm noradrenaline appearance rate was unchanged. Lower-body negative pressure did not affect the forearm noradrenaline spillover rate, but increased the forearm noradrenaline appearance rate from 3.4+/-0.4 pmol x min(-1) x 100 ml(-1) at baseline to 5.0+/-0.9 pmol x min(-1) x 100 ml(-1) (P<0.05). These results indicate that the noradrenaline appearance rate provides the better approximation of changes in forearm neuronal noradrenaline release in response to stimuli which alter local blood flow.

Adult↗
PubMed1999

Quantitative pp65-antigenemia assay for the prediction of human cytomegalovirus disease in HIV-infected patients.

OBJECTIVE: To evaluate the ability of a quantified pp65-antigenemia assay to predict the development of human cytomegalovirus (HCMV) disease in patients with an advanced HIV infection. DESIGN: A prospective longitudinal study between March 1993 and December 1996. Blood samples for the pp65-antigenemia assay were drawn at 2-3 month intervals. SETTING: AIDS department of an institutional tertiary care centre. PATIENTS: A total of 101 HIV-infected patients with CD4 lymphocyte counts of 100/mm3 or less were enrolled. Ninety-seven patients were eligible for analysis. All patients gave informed consent. MAIN OUTCOME MEASURES: The development of HCMV disease. RESULTS: Of the 97 patients, 24 developed HCMV disease after a median follow-up of 10.6 months. Three months before the development of HCMV disease, an increase in the median number of pp65-antigen-positive leukocytes was observed. The highest combination of sensitivity (45%) and specificity (94%) for the development of HCMV disease within the next 3 months was found when an assay cut-off level of 48/10(5) pp65-antigen-positive leukocytes was applied, with a positive predictive value (PPV) for the development of HCMV disease of 75%. The Kaplan-Meier estimate of HCMV disease-free survival after patients reached 48/10(5) or more antigen-positive leukocytes on longitudinal follow-up was a median 3.7 months [95% confidence interval (CI), 2.5-8.5]. The hazard ratio (HR) of this threshold level for the development of HCMV disease was 9.6 (95% CI, 4.2-21.8). CONCLUSION: Longitudinal follow-up using the pp65-antigenemia assay of HIV-infected patients with a low CD4 lymphocyte count improves the identification of patients who will develop HCMV disease in the foreseeable future, and should be considered for the selection of patients who may benefit from pre-emptive HCMV treatment.

Adult↗
PubMed1999

Molecular cloning and characterization of the mouse Ecm1 gene and its 5' regulatory sequences.

The mouse Ecm1 (extracellular matrix protein 1) gene codes for an extracellular protein of 85kDa. We have determined the primary structure of this gene and analysed 1665 bases of its 5' upstream regulatory region. The gene is approximately 5kb long and contains 11 exons. The exons range in size from 45 to 375bp, whereas the intron sizes ranges from 95 to 1115bp. All splice donor/acceptor sites conform to the GT/AG rule. The 5' upstream sequences contain a TATA-box, a CCAAT-box and an inverted CCAAT-box. We have analysed the Ecm1 regulatory elements by reporter gene constructs and transient transfections in the stromal osteogenic cell line MN7. Progressive deletion of the Ecm1 promoter revealed the presence of a region with a repressive activity between -110 and -317 and showed that a 110-bp fragment, containing potential binding sites for AP1, Sp1, GATA and Ets family of transcription factors, is sufficient for CAT expression in MN7 cells. Except for the GATA binding site, these regulatory sequences are conserved in the human promoter. Point mutation analysis revealed that the AP1, Sp1 and Ets binding sites are absolutely necessary for Ecm1 expression in MN7.

Animals↗
PubMed1999

High-cell-density phorbol ester and retinoic acid upregulate involucrin and downregulate suprabasal keratin 10 in autocrine cultures of human epidermal keratinocytes.

Autocrine growth of human epidermal keratinocytes is initiated in subconfluent cell cultures in the absence of exogenous growth factors, at low calcium concentration of the medium and with sufficient cell density. Culture confluence inhibits keratinocyte proliferation and upregulates expression of early, keratin 10 (K10), and late, involucrin, markers of differentiation. In this report, the phenotype of autocrine keratinocytes was studied at high cell density (postconfluence), specifically after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), or all-trans retinoic acid (RA). At postconfluence, K10 is decreased but not involucrin. TPA upregulates involucrin expression, but not K10 in subconfluent keratinocytes. Treatment of confluent keratinocytes with RA downregulates K10, but upregulates involucrin. This in vitro culture model, unlike others, simulates for the first time the in vivo effects of RA, a member of the retinoid family which potently modulates keratinocyte differentiation and the expression of selected gene products. It thus can be developed to further examine epidermal differentiation.

Biomarkers↗
PubMed1999

Hemodynamic changes caused by glibenclamide in isolated, working, erythrocyte perfused rat heart.

Glibenclamide-induced cardiac hemodynamic changes before and after ischemia have been frequently studied. In general a Langendorff buffer perfused heart model was used to examine these effects. However these models used protein-free buffer perfusates. To improve clinical relevance and thereby enhance extrapolation to the in vivo condition we studied the effects of glibenclamide on cardiac hemodynamics using a working, erythrocyte perfused, rat heart model, where the perfusate was enriched with albumin. The results show a dose-dependent decline in CBF in normoxia and at the end of reperfusion (after an ischemic period) with glibenclamide treatment compared to control. Cardiac functional recovery improved with 1 and 4 mumol.L-1 glibenclamide concentrations. From this study it seems that there is a marked decrease in CBF but this did not result in impaired myocardial function after a period of ischemia, so it appears that there are no startling side effects of glibenclamide in the ischemic rat heart.

Animals↗
PubMed1999

Arm position is important for blood pressure measurement.

AIM: To test the effect of positioning the arm on the arm-rest of a common chair, below the officially recommended right atrial level, on the blood pressure (BP) readings in a group of out-patients. PATIENTS AND METHODS: A group of 69 patients (58 hypertensives; 39 males; mean +/- s.d. age 54.1 +/- 16.0 years) participated in the present study. BP and heart rate values obtained in each of the following two positions were compared: (1) sitting with the arms supported on the arm-rests of the chair and (2) sitting with the arms supported at the level of the mid-sternum (the approximation of the right atrial level). BP was measured simultaneously at both arms, with a mercury sphygmomanometer at the right arm and with an automatic oscillometric device at the left arm. RESULTS: Both the systolic and diastolic BPs were significantly higher (P < 0.0001) when the arm was placed on the arm-rest of the chair than at the right atrial level. The same differences +/- s.d. in BP between the two positions were obtained with both measurement techniques: 9.7 +/- 9.4 mm Hg (systolic) and 10.8 +/- 5.8 mm Hg (diastolic) with the mercury sphygmomanometer and respectively 7.3 +/- 8.9 mm Hg and 8.3 +/- 6.0 mm Hg with the oscillometric device. No difference in the heart rate was found between the two positions. CONCLUSIONS: Placing the patient's arms on the arm-rest of the chair instead of at the reference right atrial level, BP measurement will result in spuriously elevated BP values. This may be of great importance for the diagnosis and the subsequent treatment decisions for patients with hypertension.

Adult↗
PubMed1999

The impact of blood pressure measurement methods on the assessment of differences in blood pressure levels between patients with normoalbuminuric type 1 diabetes and healthy controls.

The aim of the study was to investigate the impact of various blood pressure (BP) measurement methods on the assessment of differences in BP levels between patients with normoalbuminuric type 1 diabetes and healthy controls. We measured intra-arterial BP (i.a.), sphygmomanometric BP (sphygmo), 24-h ambulatory blood pressure (ABPM, auscultatory, Profilomat) and oscillometric BP (Dinamap) in 51 patients with normoalbuminuric type 1 diabetes (DP) with a mean diabetes duration of 8.4 years and 42 healthy controls (C). Results are expressed as mean +/- SE. There was no significant difference in i.a. BP between DP and C (systolic/diastolic BP and mean arterial pressure (MAP) 116.2 +/- 1.2/61.7 +/- 0.8 (82.8 +/- 0.9) mm Hg in DP vs 115.6 +/- 1.2/63.2 +/- 0.9 (83.4 +/- 1.1) in C). Sphygmo BP was 117.7 +/- 1.3/69.8 +/- 1.0 mm Hg in DP vs 116.5 +/- 1.5/67.8 +/- 1.3 in C (NS). Also, ABPM was not significantly different between both groups. Daytime BP between 10.00-23.00 h was 120.9 +/- 1.2/84.4 +/- 0.9 mm Hg in DP vs 120.4 +/- 1.5/83.7 +/- 1.0 in C (NS). Night-time BP between 01.00-07.00 h was 102.4 +/- 1.2/69.3 +/- 0.9 mm Hg in DP vs 103.4 +/- 1.5/69.1 +/- 1.3 in C (NS). In contrast, systolic Dinamap BP was higher in DP (118.6 +/- 1.3 in DP vs 113.4 +/- 1.4 mm Hg in C, P = 0.01) as was MAP (85.6 +/- 0.7 in DP vs 83.3 +/- 1.0 mm Hg in C, P = 0.05). Diastolic Dinamap BP was not significantly different (66.6 +/- 0.7 in DP vs 65.0 +/- 1.0 mm Hg in C). We conclude that intra-arterial BP was similar in patients with normoalbuminuric type 1 diabetes and healthy controls. Also, when using auscultatory BP devices there were no apparent differences in blood pressure. In contrast, using the oscillometric method (Dinamap), BP especially systolic, was higher in diabetic patients. Measurements with an oscillometric device (Dinamap) might therefore overestimate BP in patients with normoalbuminuric type 1 diabetes, thus confusing conclusions on the relationship between development of hypertension and microalbuminuria in the early phase of diabetes.

Adult↗
PubMed1999

Flow ratios to express results obtained with the human in vivo 'perfused forearm technique'.

AIMS: To determine the accuracy of forearm blood flow (FBF) ratio (flow in infused arm/flow in control arm) to detect unilateral increases in forearm blood flow. METHODS: In nine healthy male volunteers, we measured the effect of infusion of saline into the brachial artery at a rate of 2 ml/100 ml forearm min-1 on FBF ratio during control, mental arithmetic (MAR) and lower body negative pressure (LBNP) at -40 mmHg. RESULTS: Saline infusion increased FBF ratio from baseline by 115.9+/-17.4, 82.0+/-19.0 and 159.6+/-53.3% for control, MAR and LBNP, respectively (P<0.05 for MAR vs control). CONCLUSIONS: FBF ratio may underestimate unilateral increases in forearm blood flow during simultaneous mental arousal.

Adult↗
PubMed1999

Atrial natriuretic peptide increases albuminuria in type I diabetic patients: evidence for blockade of tubular protein reabsorption.

BACKGROUND: It has been suggested that atrial natriuretic peptide (ANP) contributes to the glomerular hyperfiltration of diabetes mellitus. Infusion of ANP increases the urinary excretion of albumin in patients with type I diabetes mellitus (IDDM). Although the increased albuminuria is attributed to a rise in glomerular pressure, alterations in tubular protein handling might be involved. PATIENTS AND METHODS: We have studied the effects of ANP in nine microalbuminuric IDDM patients. After obtaining baseline parameters, ANP was infused over a 1-h period (bolus 0.05 microgram kg-1, infusion rate 0.01 microgram kg-1 min-1). Renal haemodynamics, sodium and water clearance and tubular protein handling were studied. RESULTS: The glomerular filtration rate (GFR) increased from 116.4 +/- 8.9 to 128.3 +/- 8.8 mL min-1 1.73 m-2, whereas the effective renal plasma flow (ERPF) decreased from 534.3 +/- 44.3 to 484.9 +/- 33.3 mL min-1 1.73 m-2 (P < 0.05). As a result, the filtration fraction was significantly higher during infusion of ANP. ANP attenuated proximal tubular sodium reabsorption. Urinary albumin excretion rose from 87.57 +/- 21.03 to 291.40 +/- 67.86 micrograms min-1 (P < 0.01). Changes in the urinary excretion of beta 2-microglobulin and free kappa light chains were more marked, the excretion of beta 2-microglobulin increasing from 0.28 +/- 0.21 to 51.87 +/- 10.51 micrograms min-1 (P < 0.01), and of free kappa-light chains from 4.73 +/- 1.74 to 46.14 +/- 6.19 micrograms min-1 (P < 0.01). CONCLUSIONS: The observed rise in albuminuria during infusion of ANP does not simply reflect a change in glomerular pressure, but might at least partly result from an attenuation of tubular protein reabsorption.

Adult↗
PubMed1999

Transcapillary escape rate of albumin is increased and related to haemodynamic changes in normo-albuminuric type 1 diabetic patients.

OBJECTIVE: An increase in urinary albumin excretion (UAE) in type 1 diabetic patients might reflect changes in vascular permeability and/or local haemodynamic factors. Indeed, transcapillary escape of albumin (TERalb), a measure of systemic capillary efflux, is increased in diabetic patients, even in those with a modest increase of albuminuria. In normo-albuminuric type 1 diabetic patients, systemic capillary and glomerular flow is increased. We hypothesized that these haemodynamic changes contribute to an elevated TERalb, even in the phase preceding micro-albuminuria. METHODS: We measured TERalb in 39 normo-albuminuric type 1 diabetic patients and 46 healthy controls. TERalb was calculated from the disappearance curve of 125I-albumin. Renal and systemic haemodynamics were measured by standard techniques. Forearm blood flow (FBF) was measured by plethysmography. Endothelial function was assessed by intra-arterial infusion of acetylcholine. The structural integrity of the vessel wall was determined by the post-occlusive reactive hyperaemia test. RESULTS: TERalb was increased in diabetic patients (5.53+/-0.40 versus 4.39+/-0.21 %/h, P = 0.01). Patients were divided into tertiles with respect to their TERalb. There were no differences in UAE, blood pressure, metabolic parameters, endothelial function or maximal vasodilatation after occlusion between the groups. However, filtration fraction and FBF were significantly increased in the group of diabetic patients with the highest levels of TERalb. Overall, in diabetic patients, FBF was significantly correlated with TERalb. CONCLUSIONS: TERalb is increased in normo-albuminuric type 1 diabetic patients. In these patients with an increased capillary permeability, there is no evidence of endothelial dysfunction or vessel wall damage. However, both FBF and filtration fraction are increased. Therefore, the increased vascular permeability in the early phase of type 1 diabetes is associated with general haemodynamic alterations. Notably, such an increase in vascular permeability is not necessarily reflected by abnormal UAE. This could be due to either a lack of change in glomerular permeability or due to the fact that the threshold for tubular reabsorption of albumin has not been exceeded.

Adult↗
PubMed1999

The role of adenosine in insulin-induced vasodilation.

It was previously shown that systemic hyperinsulinemia induces vasodilation in human skeletal muscle. The mechanism mediating this vasodilation is not yet completely clarified. Based on data from animal experiments, we hypothesized that stimulation of the adenosine receptor is involved in insulin-induced vasodilation. To test this hypothesis, a 105-min hyperinsulinemic euglycemic clamp was performed in three groups of eight healthy volunteers. In group 1, placebo was infused into the left brachial artery (experimental forearm). In the second and third group, respectively, draflazine (an adenosine-uptake blocker) and theophylline (an adenosine-receptor antagonist) were administered by intrabrachial infusion. Forearm blood flow (FBF) was measured by venous-occlusion plethysmography, both at the experimental and the control forearms. The percentage decrease in flow ratio (FBF experimental arm/control arm) in the draflazine group was significantly less pronounced than that in the placebo group, whereas the percentage decrease in flow ratio was larger in the theophylline group. These results demonstrate that the insulin-induced increase in blood flow in the experimental arm was more pronounced at the site of adenosine-uptake blockade by draflazine, whereas this was reduced during adenosine-receptor antagonism by theophylline. Our observations are compatible with the hypothesis that insulin-induced vasodilation is mediated by the release of adenosine.

Adenosine↗
PubMed1999

Presence and mechanism of direct vascular effects of amiloride in humans.

Besides an evident diuretic effect, amiloride has been shown to exert direct vasoactivity in various animal experiments, whereas human data on this issue are lacking. Inhibition of Na+/H+ exchange, alpha-adrenergic blockade, and sodium and calcium channel antagonism have been proposed as possible mechanisms of this action. Although the role of Na+/H+ exchange in vascular-tone modulation is not completely clear, various vasoconstrictive agents (e.g., angiotensin II) enhance its activity. We examined the direct effects of amiloride on human arterial vasculature in vivo. Forearm vasodilator responses to the infusion of placebo and amiloride (n = 10; 0.1-100 microg/min/dl) into the brachial artery were recorded by venous occlusion strain-gauge plethysmography. Reduction of forearm blood flow after local administration of noradrenaline or angiotensin II was measured before and after local amiloride administration. Amiloride increased the ratio of the infused/ noninfused forearm blood flow at the highest dosages (10, 30, and 100 microg/min/dl with 14+/-9, 17+/-14, 58+/-23% (p = 0.002, repeated-measures analysis of variance). In contrast to noradrenaline-induced vasoconstriction, the vasoconstrictor response to angiotensin II was significantly attenuated by amiloride (p = 0.02). At high concentrations, amiloride exerts direct vasodilator activity in human arterial vasculature in vivo. This effect appears not to depend on alpha-adrenergic receptor blockade, but shows interaction with angiotensin II, an activator of Na+/ H+ exchange.

Adult↗
PubMed1999

Inhibition of carbonic anhydrase accounts for the direct vascular effects of hydrochlorothiazide.

Hydrochlorothiazide has been shown to exert direct vasodilator effects by activation of calcium-activated potassium (KCa) channels in human and guinea pig isolated resistance arteries. Since hydrochlorothiazide binds to and inhibits the enzyme carbonic anhydrase and because KCa channel activation is pH sensitive, we investigated the role of intracellular and extracellular carbonic anhydrase in the vascular effects of thiazide diuretics. Small arteries were isolated from guinea pig mesentery and studied by use of a microvascular myograph technique. In some experiments, tone and intracellular pH (pHi) were measured simultaneously with 2', 7'-bis(2-carboxyethyl)-5(6)'-carboxyfluorescein (BCECF-AM). Bendroflumethiazide, a thiazide diuretic with minimal inhibitory effects on carbonic anhydrase, had little effect on noradrenaline-induced tone (16+/-8% relaxation) compared with hydrochlorothiazide (74+/-12% relaxation). In contrast to hydrochlorothiazide, the action of bendroflumethiazide was unaffected by 100 nmol/L charybdotoxin, a selective blocker of KCa channels. All inhibitors of carbonic anhydrase relaxed noradrenaline-induced tone in a concentration-dependent manner, and this effect was blocked by charybdotoxin. Hydrochlorothiazide and the inhibitors of carbonic anhydrase failed to relax tone induced by a depolarizing potassium solution. Acetazolamide and hydrochlorothiazide increased pHi by 0.27+/-0.07 and 0.21+/-0.04, respectively, whereas bendroflumethiazide had a much smaller effect: 0.06+/-0.03. The rise in pHi induced by any agent was not inhibited by charybdotoxin. The vasorelaxant effect of hydrochlorothiazide is shared by other inhibitors of carbonic anhydrase. Inhibitors of carbonic anhydrase, but not bendroflumethiazide, cause intracellular alkalinization, which is associated with KCa channel opening. These data suggest that the vasodilator effect of thiazide diuretics results primarily from inhibition of vascular smooth muscle cell carbonic anhydrase, which results in a rise in pHI, leading to KCa channel activation and vasorelaxation.

Animals↗
PubMed1999

Elevated skeletal muscle blood flow in noncomplicated type 1 diabetes mellitus: role of nitric oxide and sympathetic tone.

Capillary hyperperfusion precedes and contributes to the occurrence of diabetic microangiopathy. Vascular tone is regulated by the balance of vasodilating and vasoconstricting factors, of which nitric oxide (NO; an endothelium dependent vasodilator) and norepinephrine (NE; a potent vasoconstrictor), respectively, are of primary importance. To investigate the role of these factors in hyperperfusion, we measured forearm blood flow (FBF) in 50 patients with noncomplicated type 1 diabetes (DP) and 50 healthy control subjects (CS) under baseline conditions and during intrabrachial infusion of N(G)-monomethyl-L-arginine (L-NMMA), an endothelium-dependent vasoconstrictor, and acetylcholine (ACh), an endothelium-dependent vasodilator. Furthermore, we determined arterial plasma NE concentration at baseline and then determined alpha-adrenergic receptor sensitivity by measuring FBF response to intra-arterially infused NE. We found that basal FBF was increased in DP (2.9+/-0.1 versus 2.0+/-0.1 mL. min(-1). dL(-1) in CS; P<0.01). L-NMMA caused a similar vasoconstriction in both groups (28.5+/-1. 7% in DP versus 31.2+/-2.2% in CS; P=NS). Maximum blood flow during infusion of ACh was not different (23.3+/-1.9 mL. min(-1). dL(-1) in DP versus 20.1+/-1.6 in CS). Arterial plasma NE concentrations were significantly decreased in DP (0.57+/-0.03 versus 0.81+/-0.05 nmol/L in CS; P<0.01). The vasoconstrictive effect of NE was increased in DP (slope log dose-response curve, 31.3+/-1.5 versus 24.3+/-1.8 in CS; P<0.01). We conclude that basal FBF is increased in noncomplicated type 1 diabetes. We found no evidence of a disturbance of basal or stimulated NO production. Arterial plasma NE concentrations are decreased in noncomplicated type 1 diabetes. This may explain the vasodilatation at baseline and the increased vascular response to intra-arterially NE.

Acetylcholine↗
PubMed1999

Prevalence of "syndrome X" features in parents of type 1 diabetic patients with or without nephropathy.

OBJECTIVE: To compare the prevalence of "syndrome X"-related parameters in parents of type 1 diabetic patients with diabetic nephropathy (DNP) to those in parents of diabetic patients without DNP. RESEARCH DESIGN AND METHODS: In this cross-sectional study, we included 50 parents of type 1 diabetic patients with DNP and 50 parents of diabetic patients without DNP. All parents were investigated in a fasting state for serum lipids including nonesterified fatty acids (NEFAs), glucose, HbA1c, plasma uric acid, fasting insulin levels, and albuminuria. Blood pressure was recorded in the supine position using an automatic device; ankle/brachial index was measured with Doppler ultrasound. Presence of cardiovascular disease was determined by a standardized questionnaire and electrocardiogram registration. Subjects without known diabetes underwent a 2-h oral glucose tolerance test. Anthropometric parameters such as BMI, waist-to-hip ratio, and percentage of body fat were measured. In addition to univariate analysis, a syndrome X score (SXS) was formulated, comprising a number of syndrome X-related biochemical, physiological, and/or anthropometric parameters. RESULTS: Univariate analysis revealed no significant differences in syndrome X parameters between parents of type 1 diabetic patients with or without DNP. Also, the composite SXS was similar in both groups. CONCLUSIONS: In this study, no differences were found in the prevalence of syndrome X features between parents of type 1 diabetic patients with DNP and parents of patients without DNP.

Adult↗
PubMed1998

Solid-phase extraction of 18beta-glycyrrhetinic acid from plasma and subsequent analysis by high-performance liquid chromatography.

A new method is described for the solid-phase extraction of 18beta-glycyrrhetinic acid from plasma or serum, with subsequent analysis by HPLC. New aspects of the method include the use of commercially available 18alpha-glycyrrhetinic acid as the internal standard and the use of a Bond Elut C2 (ethyl) extraction column, to avoid the need to use large volumes of organic solvent to elute the isolates from the columns. Separation was achieved on a Shandon Hypersil BDS C18 analytical column, with a mobile phase consisting of acetonitrile-0.02 M phosphate buffer, pH 5.7 (55:45, v/v). The column effluent was monitored at 248 nm. Compared with previous methods, the procedure is much easier to carry out, whereas the sensitivity (limit of detection, 10 ng/ml, and limit of quantitation, 50 ng/ml), the precision (0.3-6.2%) and the accuracy (97.2-101.9%) are of the same order of magnitude.

Calibration↗