¹Northern Ireland Centre for Diet and Health (NICHE), University of Ulster, Coleraine, Northern Ireland, UK

²Borax Europe Limited, Guildford, UK

Correspondence to: J M W Wallace, Northern Ireland Centre for Diet and Health, University of Ulster, Coleraine, Co. Londonderry, BT52 1SA, Northern Ireland, UK. E-mail: j.wallace@ulster.ac.uk

^aGuarantor: JMW Wallace and Dr MPA Hannon-Fletcher.

^bContributors: JMWW assisted with the design and implementation of the intervention study and was the principal author of the manuscript. MPAHF carried out the intervention study and was involved in statistically analysing the data. PJR assisted with the design and implementation of the intervention study and advised on the dietary assessment methods. WSG assisted with the haemostatic measures. JJS assisted with the design of the study and the preparation of the manuscript. SAH assisted with the design of the study and the preparation of the manuscript.

Objective: The aim of the present study was to determine whether postprandial concentrations of the active component of serine protease coagulation factor VII (VIIa) were lowered by acute boron supplementation in vivo.

Design: An acute, randomized, placebo-controlled, double blind, cross-over study.

Setting: Free-living population.

Subjects: Fifteen apparently healthy men, aged 45-65 y.

Interventions: Subjects visited the centre on two occasions, with the study days separated by a minimum of 2 weeks. Following collection of a fasting blood sample, subjects received either placebo or acute bolus of 11.6 mg boron (given as 102.6 mg sodium tetraborate decahydrate) together with a standard fat-rich meal. Blood samples were obtained at 1, 2, 4 and 6 h after the administration of the test meal, during which time subjects were at liberty to consume deionized water only. Blood samples were assayed for concentrations of insulin, glucose, lipids and boron. Measurement of the concentration of activated factor VIIa and of factor VII antigen, and of the activity of coagulation factors VII, IX and X was also carried out.

Results: Plasma boron concentrations were significantly higher following consumption of the boron supplement compared with placebo (0.124±0.02 vs 0.008±0.01 mg/l; P£0.001). There was no significant effect of acute boron supplementation on plasma insulin and glucose concentration or on blood lipid or coagulation factor profile. Factor VIIa rose significantly following consumption of the high fat meal (1.05±0.07 vs 1.26±0.07; P£0.001), but this increase was not altered by boron supplementation.

Conclusions: Results from this study suggest that acute boron supplementation (at 11.6 mg boron) does not alter the activity of factor VIIa following consumption of a high-fat meal.

Sponsorship: This work was funded by Borax Europe Ltd.

European Journal of Clinical Nutrition (2002) 56, 1102-1107. doi:10.1038/sj.ejcn.1601455

boron; serine protease; factor VIIa; postprandial; coagulation; diet

Introduction

Although an essential role for the trace element, boron, in plants has long been established (Warrington, 1923), a primary role for boron in animals has not yet been identified. Researchers have proposed a role for this non-metallic element, which possesses properties that are intermediate between metals and non-metals (Naghii & Samman, 1997), in several major physiological processes in animals. These include cognitive function (Penland, 1998) and bone formation (Chapin et al, 1998; Armstrong et al, 2000). Embryonic defects related to boron depletion have been reported for zebra fish (Rowe & Eckhert, 1999), frogs (Fort et al, 1998, 1999, 2000) and trout (Eckhert, 1998). Boron-related development defects, however, have not been observed consistently in rodent models (Lanoue et al, 1998, 1999). Despite evidence of the essentiality of boron in animals, currently there is no recommended daily allowance (RDA) for boron intake. Recently the United States Food and Nutrition Board (2001) has set a Tolerable Upper Intake level (UL) for boron of 20 mg/day, a level well above average intakes in the UK.

Boron is known to influence the in vitro activities of several classes of enzymes present in plants and animals (Hunt, 1996). One such class of enzymes is competitively inhibited through the formation of transition state analogues with boron and boric acid. Important examples of this class are the serine proteases, where boron forms a tetrahedral adduct with the active site, serine. Serine proteases participate in complex metabolic processes including blood clotting, fibrinolysis and the blood complement system at many levels. For example, one set of serine proteases, active blood coagulation factors, acts to initiate the clotting process by activating subsequent components of the cascade system by proteolytic cleavage, ultimately resulting in the deposition of a fibrin clot.

Many recent epidemiological studies have demonstrated that elevated plasma activities of blood coagulation factors are risk factors, or are associated with risk factors, for ischaemic heart disease (Woodward et al, 1997, 1998; Smith et al, 1997; Lowe et al, 1998; Feng et al, 2000; Cooper et al, 2000). While earlier studies assessed only total activities of the blood coagulation factor VII, the development of a method for the determination of factor VIIa, the active component of factor VII (Morrissey et al, 1993), facilitated direct assessment of the role of plasma FVIIa in thrombotic disorders. Factor VIIa has been reported to be elevated after a high fat meal (Miller, 1997) with similar postprandial increases in factor VIIa observed irrespective of the fatty acid composition of the meal (Larsen et al, 1997). To date, no epidemiological study has examined boron status or the possible relationship of this trace element to the risk of ischaemic heart disease (IHD). However, given in vitro evidence and animal studies showing that boron and amino acid boron conjugates are potent inhibitors of serine proteases (Kettner & Shenvi, 1984), including blood clotting factors (Knabb et al, 1992; Badimon et al, 1994; Mitchell et al, 1994), it is possible that a boron rich diet, or boron supplementation, may help to reduce the risk of thrombosis and, thereby, the risk of IHD. The objective of the present study was to determine if acute boron supplementation affected the activity of postprandial factor VIIa.

Materials and methods

Subject selection

The study was approved by the Research Ethics Committee of the University of Ulster and informed consent was obtained from each subject at the time of recruitment to the study. Initially, 31 apparently healthy men, aged 45-65 y, volunteered to participate in the intervention study. Following a lifestyle and biochemical screen, 15 volunteers satisfied the following inclusion criteria: non-smokers, with a habitually low intake of fish (n-3 polyunsaturated fatty acids have previously been shown to lower postprandial factor VIIa; Nordoy et al, 2000) and a body mass index (BMI) of between 20 and 33 kg/m². None of the volunteers was adhering to any form of special diet or taking dietary fatty acid supplements. The volunteers had no previous history of hyperlipidaemia or endocrine disease. Subjects were negative for the Arg₃₅₃Gln polymorphism in the factor VII gene. The gene coding for factor VII has five identified polymorphic sites that may associate with circulating levels of the gene product (for review see Lane & Grant, 2000). It has previously been shown that individuals who carry the allele for Gln₃₅₃ show reduced factor VII coagulant activity and that about one-third of the variation in factor VIIc between subjects is explained by this polymorphism (Humphries et al, 1994). The baseline characteristics of the subjects are presented in Table 1. Plasma glucose and full blood count were within the normal range for all subjects.

DNA procedures

Genotyping was performed for the Arg-Gln polymorphism in the following way. DNA was extracted from isolated mononuclear cells using DNAzol (Gibco Life Technologies, Paisley, UK) and the method described in detail by Chomczynski et al (1997). Enzymatic amplification of DNA was performed by polymerase chain reaction (PCR) as described previously (Green et al, 1991). Subjects with the Arg₃₅₃Gln polymorphism were excluded from the study.

Study design

The study was a randomized, placebo controlled, double blind, cross-over study. Subjects attended the Northern Ireland Centre for Diet and Health on two occasions, each following an overnight fast (12 h). On each of the two study days, subjects received a standardized monounsaturated fatty acid (MUFA)-rich test meal. The test meal consisted of 135 g white bread, 36 g strawberry jam and a milk shake composed of 40 g olive oil, 40 g dried skimmed milk powder and 40 g strawberry-flavoured milk shake mix mixed with 200 ml of water. Each test meal provided 997 kcal (4.18 MJ; Zampelas et al, 1998). In addition to the test meal, subjects received either an acute bolus of 11.6 mg boron as 102.6 mg sodium tetraborate decahydrate (Borax Europe Ltd) or a placebo given in a single capsule. This level of boron supplementation was chosen because it has been reported that this amount would be achievable by dietary means (Nielsen, 1994). Subjects consumed the boron or placebo in a random order in two study days, separated by at least 2 weeks. The subjects were requested not to change their habitual diets during the intervention and to refrain from alcohol and boron rich foods for 24 h prior to each study day. During each postprandial study period (6 h), subjects were asked to abstain from food and drinks, but were encouraged to consume de-ionized double-distilled water, which was provided to subjects.

Sample collection

Urine samples were collected by subjects for the 24 h period preceding each study day to ensure that boron intake was not significantly different prior to each investigation. Upon arrival at the centre, subjects were canulated and baseline blood samples collected. The test meal was consumed by subjects within 20 min of canulation and the subsequent postprandial blood samples were obtained at 1, 2, 4 and 6 h after consumption of the test meal. Blood for coagulation factor analysis was collected into evacuated tubes containing sodium citrate (Vacuette, Greiner Labortechnik, Germany) while blood for plasma lipid analysis was collected into lithium heparin coated evacuated tubes (Vacuette, Greiner Labortechnik). Sodium fluoride, potassium oxalate (BD Vacutainer, Oxford, UK) -anticoagulated blood was collected for measurement of plasma glucose while EDTA (Monoject, Sherwood Medical, Northern Ireland, UK) anticoagulant tubes were used to collect blood for insulin assessment. All samples were stored at 4°C until processing within 2 h of collection, and then fractionated and stored (-70°C) until analysis upon completion of the study within 6 months.

Biochemical analysis

Plasma HDL-cholesterol, cholesterol and triglyceride concentrations were assessed at 0, 1, 2, 4 and 6 h using commercially available kits (Randox Laboratories, Crumlin, UK) on the Cobas Fara autoanalyser (Roche, USA).

Plasma insulin and glucose concentration were determined at 0, 1 and 2 h using commercially available kits (DRG Instruments, Germany; Randox Laboratories). Insulin was measured using an ELISA method which employs a direct sandwich technique using microtitre strips coated with mouse monoclonal anti-insulin antibody and a peroxidase conjugated mouse monoclonal anti-insulin secondary antibody.

Total plasma activity of factor VII (VIIC), factor IX (IXC) and factor X (XC) was determined using a one-stage clotting assay with factor VII, IX or X deficient plasma (Instrumentation Laboratory Company, Lexington, MA, USA) on the ACL 100 coagulometer (Instrumentation Laboratory). The concentration of factor VII antigen (VIIag) was measured using an ELISA method (Asserachrom VII: Ag, Diagnostica Stago, Asnières, France). This assay employs a quantitative 'sandwich' enzyme immunoassay technique using a microtitre plate coated with rabbit antibodies specific for factor VII together with secondary antibodies specific for factor VII.

Factor VIIa was measured by the method of Morrissey et al (1993). Briefly, samples were thawed quickly and held at room temperature until analysis. Samples and controls were diluted 1 in 10 with factor VII-deficient plasma (Instrumentation Laboratory). Automated clotting assays were performed using the ACL 100 as follows with the instrument operating in research mode. An aliquot of the test sample (50 µl) and a 50 µl aliquot of soluble tissue factor phospholipid complex was mixed and prewarmed to 37°C in the coagulometer. Clotting was initiated by the addition of 50 µl of 25 mM CaCl₂, prewarmed to 37°C. The time taken for a clot to form was recorded by the coagulometer and the concentration of factor VIIa was quantified from the standard curve, constructed using a factor VIIa standard. The values were then multiplied 10-fold to correct for the dilution of the test samples with factor VII deficient plasma. With each run, a normal and an abnormal (low) plasma were included to assess inter-assay variability (Instrumentation Laboratory). The inter-assay CV for the normal control plasma was 4.3% and 3.3% for the abnormal control plasma.

Boron concentration in plasma and urine was determined by West Coast Analytical Service Inc. (Santa Fe Springs, CA, USA) using inductively coupled plasma mass spectrometry (ICP-MS) as described in Pahl et al (2001). The detection limit of the assay for boron was 0.01 µg/g for the plasma samples and 0.10 mg/l for the urine samples.

Statistical analysis

Results are expressed as mean±s.d. Data were analysed using analysis of variance for repeated measures with the SPSS/PC package 9.0 (SPSS Inc. Chicago, IL, USA) with supplement type as the between-subject factor. When the overall F-test for time was significant (P£0.05), comparisons between time-points were made using paired sample t-test with Bonferroni's correction for multiple comparisons.

Results

All 15 subjects recruited onto the acute intervention study completed both postprandial investigations and reported no adverse effects of the study. The baseline characteristics of subjects on entry to the study are shown in Table 1. There were no significant differences between fasting cholesterol, triglyceride, glucose, insulin, or any of the coagulation factors measured on the two postprandial study days (see Table 2).

Mean urinary boron concentrations for the 24 h urine collections prior to the two study days (0.88±0.18 and 0.80±0.12 mg/l, respectively) did not differ significantly. Table 2 shows coagulation factors, lipid profile, insulin, glucose and boron concentrations in the test subjects at baseline, and following consumption of the fat-rich meal with and without the boron bolus. Plasma boron increased significantly (P£0.001) following consumption of the acute bolus of boron compared to the placebo. Plasma boron was significantly elevated from baseline values 1 h after consumption of the boron supplement and the concentration peaked at 4 h postprandially. At 6 h postprandially, the plasma boron concentration was still significantly higher than the baseline concentration.

Plasma triglyceride concentration increased significantly following consumption of the high fat meal (P£0.001) and had not returned to baseline 6 h postprandially. Boron supplementation had no significant effect on the postprandial increase in plasma triglycerides. Plasma cholesterol, LDL cholesterol and HDL cholesterol concentration were not altered following consumption of the high-fat meal, either with or without the boron bolus. Repeated measures ANOVA showed that plasma insulin increased significantly (P£0.001) postprandially, peaking at 1 h and although concentrations started decreasing by hour 2, they had not fallen to baseline levels by hour 6. There was no significant effect of boron on postprandial plasma glucose.

There was no significant effect of postprandial time or boron supplementation on factor VIIc, factor IXc or factor Xc activity. Factor VIIag levels increased significantly (P£0.001) following consumption of the high fat meal and peaked after 4 h. There was no significant effect of boron supplementation on postprandial factor VIIag. Factor VIIa was significantly elevated (P£0.01) following the test meal and there was no significant effect of boron on this postprandial response.

Discussion

The results from the current study suggest that acute supplementation with 11.6 mg of boron, as sodium tetraborate decahydrate, given in combination with a high-fat meal, resulted in a significant increase in plasma boron concentration compared with placebo in healthy middle-aged men. This showed that boron in the supplements was bioavailable and effectively absorbed. Overall, there was a 10-fold increase in plasma boron from fasting concentrations. To our knowledge, this is the first human study reporting on the postprandial increase in plasma boron following consumption of a boron supplement alone. Fasting plasma boron concentrations were in keeping with results of previous studies which have reported on plasma boron concentrations (Ferrando & Green, 1993; Green & Ferrando, 1994; Vanhoe et al, 1995; Pahl et al, 2001). The richest food sources of boron are fruit, vegetables, pulses, legumes and nuts. Red wine is also a rich source of boron, while drinking water may also contain boron, the concentration of which is influenced by soil boron concentration (Nielsen, 1997). Subjects in the current study were asked to refrain from boron-rich foods and wine prior to each intervention. It is likely that this request was adhered to as the boron content of the 24 h urine collections was low, indicating intakes of less than 1 mg of boron per day.

It has been reported that boron excretion changes rapidly with boron intake, suggesting that the kidney is the primary tissue for regulating boron homeostasis. In this intervention trial, the level of supplementation, 11.6 mg, was chosen because it has been reported that this amount would be achievable by dietary means and was at a level considered to be safe (Nielsen, 1994), being well below the Tolerable Upper Intake Level of 20 mg boron/day recently set by the US Food and Nutrition Board (2001). An intake of 11.6 mg boron/day would, however, be considerably higher than current intakes which are reported, in the UK, to be on average 2.8 mg boron/day (Naghii & Samman, 1993), an observation attributed to our habitually low consumption of boron-rich foods.

The results from the present study, indicating a significant increase in factor VIIa activity, factor VIIag concentration and triglyceride concentration following consumption of the monounsaturated fatty acid rich meal, are consistent with observations from several previous studies (Sanders et al, 1999, 2000; Roche et al, 1998). This increase was evident on each study day with no significant effect of boron supplementation on the postprandial response in these individuals, even though the presence of postprandial boron in plasma indicated that the supplement was bioavailable. Owing to the fact that factor VIIa is a serine protease, we had proposed that boron, a serine protease inhibitor, would alter the activity of this coagulation factor. The observed lack of effect might have been owing to the acute supplementation, which may not have been sufficiently long to elicit changes in active factor VIIa. Furthermore, although subjects consumed a low-boron diet for 24 h prior to the intervention this would not have been long enough to induce boron deficiency and it is, therefore, possible that a beneficial effect of boron supplementation may only be evident in individuals who are frankly deficient in boron. Although standardized blood collection and handling procedures were used throughout the study, we cannot rule out the possibility that variations in the quality of venepuncture may have impinged on our results. Therefore, in hindsight, it may also have proved beneficial to have included assessment of fibrinopeptide A (FPA) or prothrombin activation peptide fragments (F1+F2) previously reported as an index of the quality of venepuncture (Miller et al, 1995). In conclusion, although the results from the current study show that acute supplementation at 11.6 mg with boron does not alter activated factor VII, we cannot rule out the possibility that longer-term supplementation with boron or supplementation of boron deficient individuals could have significant biological effects on factor VII activity.

Acknowledgements

The tissue factor phospholipid complex and the tissue factor standard were generous gifts from Professor Jim Morrissey, Professor of Biochemistry, University of Illinois, USA. We also wish to thank Dr DJ Northington and Dr C Jacks, West Coast Analytical Service Inc., Santa Fe Springs, California for measurement of the boron content of the biological samples. We also thank Dr C Schlekat, US Borax Inc. for reviewing the manuscript and Dr G Downing, RGD Research Inc. for assistance with the analytical samples for boron analysis. We would also like to thank our study volunteers for their participation.

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Table 1 Baseline subject characteristics and fasting lipid, glucose, boron and haemoglobin concentration

Table 2 Coagulation factors, lipid profile, insulin, glucose and boron concentration in apparently healthy men following the two fat-rich meals with and without boron supplementation