Lindsay Krall

Giada Trezzi

Jordi Garcia-Orellana

Valenti Rodella

Carl-Magnus Mörth

Per Andersson

Submarine groundwater discharge at Forsmark, Gulf of Bothnia, provided by Ra isotopes

Submarine groundwater discharge (SGD) to the Gulf of Bothnia, Baltic Sea, has been believed to be insignificant from hydrological models, yet geochemical investigations of SGD in this basin are limited. In this study, 223 Ra, 224 Ra, 226 Ra, and 228 Ra have been complemented by stable δ 18 O and δ 2 Η isotopes to characterize and quantify SGD rates from the coast of Forsmark, Sweden to Öregrundsgrepen Strait (Gulf of Bothnia). In shallow bays, SGD has been traced using relations between Ra, δ 2 Η and δ 18 Ο isotopes and salinity. Zonal SGD from deformation zones, expressed as hydraulically conductive fractures, overlain by the Börstilåsen esker, a strip of conductive gravel sediment, have been traced through offshore 224 Ra xs and 226 Ra trends. On the basis of a 224 Ra xs mass balance, the SGD flow rate ranges from (5.5 ± 3.0) • 10 3 m 3 d -1 to (950 ± 520) • 10 3 m 3 d -1 . These rates are up to two orders of magnitude higher than those determined from local hydrological models, which consider only the fresh component of SGD. From the divergence between the hydrological and 224 Ra xs models, it is inferred that the site is influenced by a component of recirculated seawater.

Introduction

Submarine groundwater discharge (SGD) is defined as the flow of water across the sea floor, including the outward flow of fresh meteoric groundwater and the recirculation of seawater through the coastal aquifer [START_REF] Burnett | Groundwater and pore water inputs to the coastal zone[END_REF][START_REF] Moore | The effect of submarine groundwater discharge on the ocean[END_REF]. In several areas of the world, this process has been recognized as an important pathway for the transport of chemical compounds, including: nutrients (e.g. [START_REF] Rodellas | The influence of sediment sources on radium-derived estimates of submarine groundwater discharge[END_REF], metals (e.g. Windom et al., 2006;Trezzi et al., 2016), pollutants (e.g. [START_REF] Boehm | Groundwater discharge: Potential association with fecal indicator bacteria in the surf zone[END_REF], and natural radionuclides (e.g. Garcia-Orellana et al., 2013).

The Ra isotope quartet (long-lived 226 Ra and 228 Ra and short-lived 223 Ra and 224 Ra; t 1/2 ~1600 y, 5. 75 y, 11.4 d, and 3.66 d, respectively) has been used to trace and quantify the flow of SGD in several environments, particularly the Pacific, Atlantic, and Mediterranean coasts [START_REF] Swarzenski | A Geochemical and Geophysical Examination of Submarine Groundwater Discharge and Associated Nutrient Loading Estimates into Lynch Cove, Hood Canal[END_REF][START_REF] Swarzenski | Examining coastal exchange processes within a sandy beach using geochemical tracers, seepage meters and electrical resistivity[END_REF]Kwon et al., 2014 and references therein). Through application of 228 Ra, [START_REF] Moore | Submarine groundwater discharge revealed by 228 Ra distribution in the upper Atlantic Ocean[END_REF] determined that the rate of SGD to the upper Atlantic Ocean was similar to that freshwater inputs by rivers. Similarly, [START_REF] Kwon | Global estimate of submarine groundwater discharge based on an observationally constrained radium isotope model[END_REF] developed a 228 Ra-based model to estimate the rate of SGD to the Atlantic and Indo-Pacific Oceans and showed that SGD was a major source of nutrients to the oceans.

However, a noted source of uncertainty in the model was sparsity of 228 Ra observations from some areas, for instance the boreal regions, of the global ocean.

From a 226 Ra model, [START_REF] Jarsjö | Spatial distribution of unmonitored inland water flows to the sea[END_REF] suggested that the discharge of fresh groundwater accounts for ~4% of the total freshwater inflow to the boreal Baltic Sea. Nevertheless, using natural geochemical tracers off the coast of Poland, [START_REF] Symczycha | The Role of Submarine Groundwater Discharge as Material Source to the Baltic Sea[END_REF] determined that SGD processes can impact material budgets in the Baltic Sea even alongside low flow rates of fresh SGD. Little is known of SGD-driven material fluxes to the northern and eastern coasts of the Baltic Sea. However, they have been presumed to be less important than to the southern and eastern Baltic coasts [START_REF] Symczycha | The Role of Submarine Groundwater Discharge as Material Source to the Baltic Sea[END_REF].

Ra isotope methodology is typically applied in saline water due to the potential for Ra adsorption to particles in low salinity environments [START_REF] Li | Desorption of Ba and 226 Ra from river-borne sediments in the Hudson estuary[END_REF][START_REF] Elsinger | 226 Ra behavior in the Pee Dee River-Winyah Bay estuary[END_REF]. However, long-and short-lived Ra isotopes may allow various components of SGD to be discriminated, including relative contributions from fresh groundwater and recirculated seawater (e.g. [START_REF] Charette | Uranium-and thorium-series nuclides as tracers of submarine groundwater discharge[END_REF][START_REF] Rodellas | Using the radium quartet to quantify submarine groundwater discharge and porewater exchange[END_REF]. Therefore, despite the brackish salinity of the Baltic Sea, it is of interest to characterize SGD to the Gulf of Bothnia using Ra isotopes.

Forsmark (Sweden) lies on the southwest coast of the Gulf of Bothnia, the northern-most basin of the Baltic Sea (Figure 1.a). As a current host of a geologic repository for low-level radioactive waste and a proposed host of a high-level waste repository, the hydrology and geochemistry around Forsmark have been thoroughly investigated [START_REF] Skb | Site description of Forsmark at completion of the site investigation phase-SDM-Site Forsmark[END_REF]. This provides an ideal backdrop to apply Ra isotopes to quantify and characterize SGD in a brackish environment. δ 18 O, δ 2 Η and major cation measurements complement the 224 Ra xsbased SGD flow estimates. The purpose of this study is to characterize and quantify SGD from Forsmark to the Gulf of Bothnia through integration of the hydrological knowledge of the site with geochemical and isotopic tracers.

Site background

Baltic seawater (hereafter "brackish water") is brackish due to mixing between fresh water sourced from the Scandinavian mainland and saline water from the north Atlantic Ocean. The salinity ranges from one in the northern Gulf of Bothnia to nine near the Danish straits. Since seawater exchange through the Danish straits is limited, tidal sea level fluctuations are minor throughout the Baltic Sea (Voipio, 1981). [START_REF] Symczycha | The Role of Submarine Groundwater Discharge as Material Source to the Baltic Sea[END_REF] have suggested that groundwater discharge to the Baltic Se occurs mainly along the southern and eastern coasts. Through geochemical methods, the rate of fresh SGD to the Bay of Puck, along the Polish coast, was determined to range from 9 • 10 -4 m 3 m -2 d -1 to 2.8 • 10 -2 m 3 m -2 d -1 and was correlated with monthly precipitation. The recirculated seawater component, with a rate ranging from 4 • 10 -3 to 4 • 10 -2 m 3 m -2 d -1 was found to be important [START_REF] Symczycha | The Role of Submarine Groundwater Discharge as Material Source to the Baltic Sea[END_REF].

Forsmark, Sweden lies along the coast of the Öregrundsgrepen strait, within the Sub-Cambrian Peneplain, a widespread, low-relief landscape within Fennoscandia. The ~6 x 4 km 2 area investigated for the repositories reaches a maximum elevation of ~20 m.a.s.l. Several hydraulically conductive fracture zones intersect the bedrock surface of Öregrundsgrepen, through which the discharge of brackish-to-saline groundwater to the strait is possible (Laaksoharju et al., 2008, Figure 1.b. and 1.c). [START_REF] Jarsjö | Spatial distribution of unmonitored inland water flows to the sea[END_REF] developed a hydraulic model for the Forsmark site and attributed up to 20% of the total freshwater input to Öregrundsgrepen (2.4 • 10 3 m 3 d -1 or ca. 1.5 • 10 -4 m 3 m -2 d -1 ) to fresh SGD from diffuse sources along the coast.

Deposition of glacial sediments, such as glacial sand and clay, both with low hydraulic conductivities, occurred throughout the Forsmark land and seabed during the Pleistocene glaciations [START_REF] Hedenström | Depth and stratigraphy of regolith at Forsmark, site descriptive modelling Forsmark[END_REF]. The Börstilåsen esker, a strip of hydraulically conductive gravel sediments deposited directly over the bedrock surface, extends NW-SE across the land and seabed (Figure 1.a). These deposits are overlain by an Fe,Mn-(oxyhydr)oxide-bearing mud (Borgiel, 2004;Voipio, 1981).

Although SGD to the Swedish coast of the Baltic Sea has been presumed low, pockmarks related to permeable layers in glacial clay deposited on the seabed in the Stockholm archipelago, approximately 200 km from Forsmark, have been observed by [START_REF] Söderberg | Stratabound submarine terraces and pockmarks-indicators of spring sapping in glacial clay[END_REF] and [START_REF] Jakobsson | Seafloor terraces and semicircular depressions related to fluid discharge in Stockholm Archipelago, Baltic Sea[END_REF]. These formations mark an area of gas (Hovland, 1985) or groundwater escape (Robb 1984) and in this region range from centimeters to tens of meters in size (Söderberg & Floden, 1997;[START_REF] Jakobsson | Seafloor terraces and semicircular depressions related to fluid discharge in Stockholm Archipelago, Baltic Sea[END_REF]. Limited geochemical work has been performed alongside the geophysical documentation of pockmarks along the Swedish coast. However, pockmarks on the Baltic coast of Germany were associated with an SGD rate of 9 • 10 -3 m 3 m -2 d -1 [START_REF] Schlüter | Spatial distribution and budget for submarine groundwater discharge in Eckernförde Bay (Western Baltic Sea)[END_REF]. [START_REF] Symczycha | The Role of Submarine Groundwater Discharge as Material Source to the Baltic Sea[END_REF] suggest that discharge to the Baltic coast from glacial sediments has been underestimated.

Sampling and methods

Field surveys

Three sampling campaigns were performed at Forsmark, in the strait of Öregrundsgrepen, to collect water and sediment samples for Ra and geochemical analysis. In November 2013, three 25 L samples of seawater were taken directly from the Forsmark shore (S1-S3) and salinities were measured using a YSI-30 probe.

A larger campaign in spring 2014 included the collection of water samples from: three fivestation transects (t1-5, t6-10, and t11-15), two stations in the shallow bay Tixelfjärden (TF1-2), four stations in the larger bay Kallrigafjärden (KF1-4), and five piezometers which represent shallow groundwater (SFM0001, SFM0032, SFM0049, SFM0059, SFM0060; ~5 m.b.s.l.). Additionally, deeper groundwater, representative of two of the fracture zones which intersect the bedrock surface of Öregrundsgrepen, was sampled from two boreholes (KFR01, KFR07A; ~50 m.b.s.l.) (Figure 1.a, 1.b). At each sea station, 50 L samples for Ra analysis and 0.5 L samples for δ 18 O, δ 2 H, and major cation analysis, respectively, were filtered through a 0.45 µm filter. Salinity profiles were recorded in the field at each station using a YSI-650 probe. Ten-liter groundwater samples were collected for Ra analyses, and 0.5 L for analyses of δ 18 O, δ 2 H, and major cations.

In October 2014, a sediment core was collected from Tixelfjärden bay using a Van Veen grab sampler in order to conduct a Ra diffusion experiment following the methodology used by [START_REF] Rodellas | Quantifying groundwater discharge from different sources into a Mediterranean wetland by using 222 Rn and Ra isotopes[END_REF]. Fifty liters of seawater overlying the sediment core was also collected for use in the experiment.

Methods

Radium

On site, brackish water and groundwater samples were passed through columns loaded with 25 g of manganese-impregnated acrylic fiber (hereafter 'Mn-fiber') at a flow rate <1 L min -1 to extract the Ra isotopes [START_REF] Moore | Extraction of radium from natural waters using manganese impregnated acrylic fibers[END_REF][START_REF] Moore | Radium isotopes in coastal waters on the Amazon shelf[END_REF]. Two water samples were filtered through a series of two columns to check the efficiency of Ra extraction with Mn-fibers. After the Ra extraction, the Mn-fibers were rinsed with Ra-free deionized water and then partially dried [START_REF] Sun | The effects of water content and Mn-fiber surface conditions on measurement by emanation[END_REF]. The short-lived Ra isotopes ( 223 Ra and 224 Ra) were measured within seven and three days of collection, respectively, using a Radium Delayed

Coincidence Counter (RaDeCC) as described by [START_REF] Moore | Measurement of 223 Ra and 224 Ra in coastal waters using a delayed coincidence counter[END_REF]. 224 After every 20 samples an internal control were analyzed, which was also used to drift correct data if necessary.

Diffusive flux experiments

Quantification of the Ra diffusive flux from the marine sediments was performed in order to check to contribution from of Ra from bottom sediments to the Ra inventory in seawater. The sediment core and 3 L of the brackish water collected from Tixelfjärden were placed in a plastic container and the sediments were allowed to settle. Brackish water above the core was circulated through a Mn-fiber overnight, to ensure that the water was free of Ra at the beginning of the experiment. A closed loop system was assembled using a peristaltic pump and plastic tubing, interconnecting the water overlying the core to a column containing Mnfiber. Water continuously circulated through the tubing and the Mn-fiber. Fibers were replaced after progressively longer time periods (12, 24, 48, and 72 hours). Diffusive fluxes for each Ra isotope were estimated from the slope of the Ra activity versus incubation time regression line, divided by the core surface area plotted against incubation time to obtain fluxes in dpm m -2 h -1 following the methodology of [START_REF] Rodellas | Quantifying groundwater discharge from different sources into a Mediterranean wetland by using 222 Rn and Ra isotopes[END_REF].

Salinity

In the transects (stations t1-15, 1 m depth) salinity ranged between 4.63 and 4.82. The salinity in Tixelfjärden bay (TF1-2) was slightly higher than in the offshore transects (4.83-4.84). In Kallrigafjärden bay, the salinity was lower near the streams of Forsmarksån and Olandsån

(1.7, KF1) and increased toward the opening of the bay (4.7, KF4).

Salinity variation throughout the water column was within the uncertainty of the measurement (± 0.1), thus stations of < 10 m depth exhibited no significant change in salinity with depth (Figure 2). Stations with a deeper water column showed a halocline at ~10 m depth, where salinities decreased from ~4.8 to ~4.6 (Figure 2).

Shallow groundwaters were fresh, with a salinity range 0.4 to 1.3. However, Cl - concentrations in the deep groundwaters were up to a factor of three greater than brackish Baltic water, thus the deep groundwaters were more saline than the Baltic water.

Ra isotopes

In the transects, 226 Ra and 228 Ra activities ranged 10 -18 and 15 -18 dpm 100L -1 , respectively (Figure 3.a-b). These values are similar to those reported from the central Gulf of Bothnia, of 13.3 ± 1.2 and 14.7 ± 1.6 dpm 100L -1 , respectively [START_REF] Porcelli | Transport of U-and Thseries nuclides in a Baltic Shield watershed and the Baltic Sea[END_REF]. The Gradients were observed within the first 2 km offshore for the short-lived isotopes, particularly 224 Ra xs in transect t1-5 but were not observed for the long-lived isotopes.

In Kallrigafjärden bay, 223

δ 2 H and δ 18 O values

Shallow groundwaters were distinct from, while deep groundwaters were similar to, the brackish water with respect to δ 2 H and δ 18 O values (Table 1). This is because the δ 2 H and δ 18 O signature of the deep Forsmark groundwaters was imparted by a component of Littorina Sea (7000 -9000 BP predecessor to the Baltic Sea), whereas the shallow groundwater signature was imparted by current meteoric waters (-89.8 to -80.5‰ δ 2 H and -12.6 to -11.1‰ δ 18 O). δ 2 H and δ 18 O in the transects ranged -60.9 to -59.9‰ δ 2 H and -8.0 to -7.7‰ δ 18 O, respectively. A mixing line between the Forsmark data and results from the central Baltic Sea [START_REF] Fröhlich | Deuterium and oxygen-18 in the Baltic Sea[END_REF]) as an offshore end-member, deviate from the local meteoric water line due to the increased influence of evaporation in the open Baltic (Figure 4).

Brackish water in

Geochemistry

Geochemical results (Na, Ca, Ca/Mg, and Ba) are presented in Table 1. Ba exhibits chemical behavior similar to Ra due to size and ionic radius. Ca/Mg ratios can be used to trace mixing between groundwaters and brackish water, as Ca in groundwater is predominantly sourced by aquifer solids whereas Mg by the brackish water [START_REF] Smellie | Explorative analysis of major components and isotopes-SDM-Site Forsmark[END_REF]. Therefore, geochemistry may be used to investigate Ra geochemistry and mixing between ground-and brackish Baltic water. Ba concentrations ranged 15 -20 µg L -1 in the brackish water, 21 -73 µg L -1 in the shallow groundwater, and 33 -51 µg L -1 in the deep groundwater. However, Ca is more enriched than Ba in groundwater relative to seawater. Deep groundwater concentrations are particularly high, ~530 mg Ca L -1 , while the shallow groundwater concentration (~95 mg Ca L -1 ) is only slightly higher than the brackish Baltic water (~65 mg Ca L -1 ). Groundwater Ca/Mg ratios are over an order of magnitude higher than brackish water ratios.

Ra diffusion from sediments

Sediment diffusion experiment results were used to derive the flux of Ra diffusion from the fine-grained, Fe,Mn (oxyhydr)oxide-bearing sediments deposited over the Öregrundsgrepen seabed. Calculated 223 Ra and 224 Ra xs diffusion rates amount to (2.2 ± 0.02) and (32 ± 2) dpm m -2 d -1 , respectively (Figure 5). These represent upper limits because during the experiment Ra was continuously removed from the water overlying the sediment core, thereby forcing diffusion by maximizing the gradient between the porewater and the overlying water.

Discussion

Geochemical behavior of Ra

Ra exhibits expected salinity-dependent ion exchange behavior, where Ra in fresh water is predominantly adsorbed to particles. When these particles are transported to saline water, the Ra adsorbed to the particles desorbs after exchange for cations that are enriched in seawater relative to fresh water, particularly Na. Thus, Ra isotopes may lead to an underestimation of SGD if utilized in fresh waters, in which Ra is likely by adsorption attached to particles.

The salinity after which Ra behaves conservatively is unclear. [START_REF] Gonneea | New perspectives on radium behavior within a subterranean estuary[END_REF] [START_REF] Kiperwas | Radium Isotopes as Tracers of Groundwater-Surface Water Interactions in Inland Environments[END_REF]. In Öregrundsgrepen, the salinity does not exceed five.

Since only the study by Raanan [START_REF] Kiperwas | Radium Isotopes as Tracers of Groundwater-Surface Water Interactions in Inland Environments[END_REF] supports conservative behavior of Ra at salinities less than five, it should be examined whether Ra is an appropriate tracer to apply in the brackish Öregrundsgrepen water.

In seawater, Ra desorption is promoted because it is outcompeted by Na for exchange sites, the latter of which is several orders of magnitude more enriched in seawater than in freshwater. Since exchange of Ra for Na is a heterovalent cation exchange process, the reaction involves exponential terms and is influenced by total ionic strength of the solution.

Nevertheless, an ion exchange model can be used to understand competition between monovalent and divalent cations for adsorption sites at a range of salinities, assuming that Ra sorption is controlled by an ion exchange rather than a surface complexation mechanism.

Equilibrium between a monovalent and divalent cation is expressed by the mass action equation ( 1):

! !"!!"#$ ! !"!!"#$ ! = ! ! !" !! ! !" ! ! (1)
where X represents the fractional equivalents of the mono-or divalent cation in the solid phase, m the dissolved molar concentrations, and K the composition-dependent selectivity coefficient of the particle. Here, the monovalent cations are represented by Na concentrations in freshwater, brackish Baltic water, and seawater because Na is the most abundant cation in brackish and seawater. Divalent cations are represented by concentrations of Ca, which is more similar to Ra than Mg in size and therefore exhibits more comparable ion exchange behavior.

Ca should not exceed 10% of the total Na and Ca adsorbed in brackish water unless K(Na/Ca) exceeds 200 (Figure 6). For reference, K(Na/Ca) values for montmorillonite, a clay with strong adsorptive properties, range from 2.5 to 6 depending on the ratio of solid to water (25 g L -1 and 2.5 g L -1 , respectively; Tertre et al., 2011). Through comparison between these selectivity coefficients, it is inferred that brackish water is more similar to seawater than to fresh water with respect to competition between monovalent and divalent cations. Since adsorption of Ca to particles is not significant in brackish water and the ion exchange behavior of Ra is comparable to that of Ca due to size and ionic radius, adsorption of the Ra to particles should not hinder its application in these brackish waters.

Figure 6: Fractional occupancy of Ca on solid phase for seawater, brackish water, and fresh water as a function of selectivity coefficient (K).

End-member mixing

To determine an accurate SGD flow rate, an appropriate endmember groundwater Ra activity should be selected. It would be particularly valuable to distinguish between the discharge of deep and shallow groundwater, because the former were found to contain Ra in activities up to two orders of magnitude higher than the latter. Salinity, stable δ 2 H and δ 18 O values, and

228 Ra/ 226 Ra activity ratios (ARs) have been used to define endmember groundwater components during SGD investigations in the Mediterranean [START_REF] Povinec | Characterisation of submarine groundwater discharge offshore south-eastern Sicily[END_REF][START_REF] Garcia-Solsona | Groundwater and nutrient discharge through karstic coastal springs (Castello, Spain)[END_REF][START_REF] Rodellas | Submarine groundwater discharge as a source of nutrients and trace metals in a Mediterranean bay (Palma Beach, Balearic Islands)[END_REF], while Ca/Mg ratios are potentially useful at Forsmark (Section 4.4).

228 Ra/ 226 Ra ARs in the Forsmark groundwaters (0.75 -2.40) do not differ substantially from those in the coastal and offshore brackish Baltic water (0.84 -2.07; Figure 7.a). ARs in Tixelfjärden are slightly higher than in the transects, but whether this is due to the discharge of deep or shallow groundwater cannot be distinguished from these data (Figure 7.a). Overall, Despite a salinity increase to > 4 as compared to 1.7 near the outlet of the streams (KF1), Ra desorption from suspended riverine particles would not explain the deviation in δ 2 H and δ 18 O.

Therefore, as in Tixelfjärden bay, SGD to Kallrigafjärden bay is a plausible source of Ra.

J sed + J SGD = J sea + J λ (2)
where J sed and J SGD denote the input fluxes of a specific Ra isotope through diffusion from seabed sediments and from SGD, respectively while J sea and J λ the output fluxes through

export to the open Baltic Sea and radioactive decay. J sed can be constrained using the results of the sediment diffusion experiment (Section 4.5). To constrain J sea requires determination of the excess inventory of the Ra isotope and the residence time of seawater within the investigated area. J λ is simply the mean excess Ra activity observed on the site multiplied by the decay constant. Resulting fluxes are in units of dpm d -1 .

Since a 224 Ra xs gradient was observed and more stations were enriched in 224 Ra xs than in 223 Ra, 224 Ra xs activities will be used in the mass balance. A mass balance will not be constructed using 226 Ra and 228 Ra because the average activities found in Öregrundsgrepen (13.7 and 16.0 dpm 100L -1 ) were not statistically higher than the respective activities reported from the open Baltic Sea (13.3 ± 1.2 and 14.7 ± 1.6 dpm 100L -1 ; [START_REF] Porcelli | Transport of U-and Thseries nuclides in a Baltic Shield watershed and the Baltic Sea[END_REF].

Contribution of 224 Ra from rivers and precipitation are neglected. 228 Ra activities of ~ 4 dpm 100L -1 were found at various locations in the Kalix River, which discharges into the northern Gulf of Bothnia [START_REF] Porcelli | Transport of U-and Thseries nuclides in a Baltic Shield watershed and the Baltic Sea[END_REF]. Assuming secular equilibrium between 224 Ra and 228 Ra, streams would supply only ~10% of the 224 Ra xs than the average shallow groundwater (~140 dpm 224 Ra xs 100L -1 ) could supply if shallow SGD comprises 17% of the total runoff from the Forsmark coast (Section 2).

Site constraints

To develop the mass balance, the surface area and volume of seawater affected by SGD must be constrained. A conservative estimate of the 224 (3) where i denotes the initial end-member Ra activity and obs the average 224 Ra ex / 228 Ra ex activity ratio observed within the constrained site. The subscript "ex" denotes the observed coastal 224 Ra xs or 228 Ra activity subtracted by the offshore activity (i.e. the Ra signal corrected for mixing with open seawater). An offshore 228 Ra activity of 14.7 dpm 100L -1 , reported by [START_REF] Porcelli | Transport of U-and Thseries nuclides in a Baltic Shield watershed and the Baltic Sea[END_REF] from the central Gulf of Bothnia, was used to derive 228 Ra ex , the mean of which was 1.6 dpm 100L -1 . ARs involving 224 Ra are used because the residence time is likely on the order of days, considering the size of the investigation site. This model relies on the assumption that Ra is removed from the system only through radioactive decay and exchange with open-sea waters. The initial end-member activity, i, will depend on the amount of 224 Ra xs and 228 Ra supplied to the seawater by SGD and sediment diffusion. Since this term is unknown, the highest 224 Ra ex / 228 Ra ex AR of (5.9, t1) is here used under the assumption that this ratio is found closest to the Ra source so that minimum decay is expected.

A residence time of 12 ± 14 days was obtained using 224 Ra ex / 228 Ra ex ARs of the constrained site. The uncertainty is large due to the propagation of analytical uncertainty combined with low Ra activities. However, the Ra-based T R model agrees with previous hydrological models, which determined a residence time of 9-11 days for the spring season (Engqvist, 1999). Provided the lower uncertainty of the hydrological model, a residence time of 11 ± 2 days is used to reduce the uncertainty propagated to the mass balance calculation.

Contribution from sediments

The experimental 224 Ra xs diffusive flux (32 ± 1.8 dpm m -2 d -1 ; Section 4.3.3) can be converted to total 224 Ra xs flux from sediments by multiplying them by the surface sediment area of the investigation site (16•10 6 m 2 ), under the assumption that the core sample represents sediments homogeneously distributed across the seabed of the constrained site. The resulting 224 Ra xs flux from sediment diffusion (J sed ) is (520 ± 31) • 10 6 dpm d -1 .

Selection of SGD end-member

At Forsmark, salinity, δ 2 H, Ca/Mg ratios, and 228 Ra/ 226 Ra ARs can neither be used to define the groundwater source of SGD nor to determine whether the deep or shallow groundwater is discharging (Section 5.3). However, the groundwater end-member can be selected based on the hydrological understanding of the site. For instance, the SFM0059 is a plausible shallow groundwater end-member because it is sampled from the Börstilåsen esker, a potential SGD channel (Section 2). This shallow groundwater probably mixes with some deep groundwater component sourced by the fracture network. To constrain a minimum rate of SGD, the shallow and deep groundwaters which had the highest 224 Ra xs activities (SFM0059 and KFR01, respectively) are used in the SGD rate calculation.

SGD rate estimation

The coastal 224 Ra xs inventory, or the total activity of 224 Ra xs in the volume of brackish water influenced by SGD (Section 4.1.1), must be established for the mass balance (Equation 2).

The inventory is determined by 1) multiplying the 224 Ra xs activities of the stations within the constrained site by respective water column depth, 2) averaging the results of (1) (in dpm m - 2 ), and 3) multiplying (2) by surface area of the constrained site (16•10 6 m 2 ). The resulting inventory, (4800 ± 1700) • 10 6 dpm, can then be apportioned into J sea , J sed , J λ , and J SGD (in dpm d -1 ). Rearranging Equation 2 to J SGD = J sea + J λ -J sed yields an SGD-derived 224 Ra xs flux of (640 ± 350) • 10 6 dpm d -1 . The flow rate is obtained by dividing this flux with the 224 Ra xs activity of the end-member groundwaters (Table 2). The resulting SGD rate ranges from (5.5 ± 3.0) • 10 3 to (950 ± 520) • 10 3 m 3 d -1 (or 3.4 • 10 -4 to 5.8 • 10 -2 m 3 m -2 d -1 ) depending on whether the end-member is comprised of entirely deep or shallow groundwater, respectively. In reality, SGD is probably comprised of a mixture between shallow and deep groundwater, thus an SGD flow between the two estimates is expected. Proportions deep and shallow groundwater contribution the coastal has not been determined.

Recirculated component of SGD

Previous hydrological models of the Forsmark area determined a shallow fresh groundwater discharge rate of ~2.4 • 10 3 m 3 d -1 or 1.5 • 10 -4 m 3 m -2 d -1 [START_REF] Jarsjö | Spatial distribution of unmonitored inland water flows to the sea[END_REF] Tidal cycles [START_REF] Robinson | Effect of tidal forcing on a subterranean estuary[END_REF], geothermal convection (Wilson, 2005), waves [START_REF] Robinson | Effect of tidal forcing on a subterranean estuary[END_REF], currents and irregular bathymetry (Thibodeaux & Boyle, 1987) have been shown to support seawater recirculation. Although tidal cycles are insignificant in the Baltic Sea, the mean sea level change in Öregrundsgrepen is 0.02 ± 0.2 m d -1 but can change by up to 1.3 m d -1 as a result of changes in atmospheric pressure and wind [START_REF] Aquilonius | The marine ecosystems at Forsmark and Laxemar-Simpevarp. SR-Site Biosphere[END_REF]. The wind speed in the strait is typically higher than 6.5 m s -1 and is frequently higher than 10.5 m s -1 [START_REF] Larsson-Mccann | Meteorological, hydrological and oceanographical information and data for site investigation program in the communities of Östhammar and Tierp in the northern part of Uppland[END_REF]. Geothermal convection may also drive seawater recirculation in this region, as [START_REF] Söderberg | Stratabound submarine terraces and pockmarks-indicators of spring sapping in glacial clay[END_REF] observed contrasting temperatures between warm water seeping from seafloor eskers and cold seawater during early spring in the Stockholm archipelago, 200 km south of Öregrundsgrepen. Finally, the entire Baltic Sea basin has developed irregular bathymetry through glacial erosion and sediment deposition (Voipio, 1981).

The rate of fresh SGD to Öregrundsgrepen (1.5 • 10 -4 m 3 m -2 d -1 ; [START_REF] Jarsjö | Spatial distribution of unmonitored inland water flows to the sea[END_REF] is lower than the SGD rates found in the Bay of Puck, Poland (up to 2.8 • 10 -2 m 3 m -2 d -1 ) and Eckernförde Bay, Germany (9 • 10 -3 m 3 m -2 d -1 ) [START_REF] Symczycha | The Role of Submarine Groundwater Discharge as Material Source to the Baltic Sea[END_REF]Schlüter et al., 2004, respectively), which are the only two locations in the Baltic Sea where geochemical studies of SGD have been conducted. Consideration of a recirculated component in Öregrundsgrepen increases the SGD rate to 5.8 • 10 -2 m 3 m -2 d -1 , which is higher than that reported from the southern Baltic. While the importance of a recirculated component in delivering nutrients and metals to the Baltic Sea is yet unknown, recirculated seawater is an important source of nutrients to the coastal ocean (Weinstein et al., 2011). Accurate determination of SGD-driven nutrient fluxes to the Baltic Sea represents an important area of future research. Finally, it will be valuable to further understand the impact of geothermal convection, irregular bathymetry, and fluctuations in atmospheric pressure on seawater recirculation, an important route of nutrient transport even in the absence of inland hydraulic head.

Conclusions

This study has shown that Ra isotopes can provide estimates of residence time and SGD flow rate at salinities less than five. Ra and stable isotopes have also been used to identify zones of groundwater discharge related to geologic features. Diffusion from seabed sediment is a source of 223 Ra and 224 Ra xs to the Gulf of Bothnia, amounting to ~10% of the total 224 Ra xs inventory in Öregrundsgrepen.

The 224 Ra xs -derived SGD flow rate ranged between (5.5 ± 3.0) • 10 3 and (950 ± 520) • 10 3 m 3 d -1 . The precise rate depends on the relative contributions of deep and shallow groundwater, which are unknown. Nevertheless, these rates are higher than would be expected from δ 2 H, δ 18 O, and Ca/Mg ratio mixing calculations. Therefore, a component of recirculated brackish water is significant in Öregrundsgrepen despite a modest, inland hydraulic head. Since this is a potential source of metals and nutrients to the coast, these findings have important implications on mass transport to the Baltic Sea and other low-relief coastal terrains.

Furthermore, hydrological models of the Forsmark site, constructed to understand radionuclide transport from the existing and planned radioactive waste repositories, have not considered a recirculated brackish water component in Öregrundsgrepen. If this can be considered a near-surface process, the effects on radionuclide transport at Forsmark is probably small. Nevertheless, the impact of recirculated seawater should not be overlooked, because coastal regions as prominent locations for nuclear power plants (World Nuclear Association, 2015), are becoming attractive hosts for radioactive waste repositories. Clearly, Ra isotope analysis is a viable and important complement to the models of sites proposed to host repositories.

Figure 1 :

 1 Figure 1: (a) Topographic map of Sweden, including Forsmark and the Gulf of Bothnia in the Baltic Sea and sampling stations in Forsmark and Öregrundsgrepen, with deformation zones, and Börstilåsen esker. (b) Conceptual illustration of Forsmark fracture network, notable geological formations, groundwater flow (narrow, solid black arrows), and potential Ra supply and removal processes (box model), modified after Follin et al. (2007).

Figure 2 :

 2 Figure 2: Salinity profiles for stations t1 (dashed), t3 (solid), and t5 (dotted).

223

  Ra and 224 Ra xs activities ranged 0.19 -0.70 and 1.0 -8.1 dpm 100L -1 , respectively (Figure3.c-d). Maximum and minimum activities were found at station t15 and t1, respectively.

  Tixelfjärden bay was enriched in heavy isotopes (maximum -58.8‰ δ 2 H and -7.59‰ δ 18 O) relative to the transects, suggesting a local influence of evaporation, which can produce this heavy signature. Application of the model byFrölich et al. (1988) to the Forsmark coast indicates that the rate of evaporation from Tixelfjärden bay is 15% of the rate of water inflow from Öregrundsgrepen to Tixelfjärden bay. This is corroborated by[START_REF] Engqvist | [END_REF], who demonstrated a faster rejuvenation of water towards the center of the strait.The lighter signature (lower isotope values) in Kallrigafjärden bay is reminiscent of influence by current meteoric water.

Figure 3 .

 3 Figure 3.a-.d: 226 Ra and 228 Ra (a-b) and 224 Ra xs and 223 Ra (c-d) activities as a function of distance offshore (m). Circle to indicate systematic increases in 224 Ra xs and 226 Ra activity.

Figure 4 :

 4 Figure 4: δ 2 H and δ 18 O of groundwater and seawater samples, samples of Fröhlich (1988), and the local meteoric water line (LMWL; Tröjbom et al., 2007).

  Diffusion-sourced226 Ra and228 Ra fluxes are locally insignificant because the rate of their production by Th parents is slow[START_REF] Rodellas | The influence of sediment sources on radium-derived estimates of submarine groundwater discharge[END_REF] relative to the residence time within Öregrundsgrepen. The 224 Ra xs diffusion rate is similar to that reported from the Venice Lagoon (32 dpm d -1 m -2 ;[START_REF] Garcia-Solsona | Estimating submarine groundwater discharge around Isola La Cura, northern Venice Lagoon (Italy), by using the radium quartet[END_REF] and Jamaica Bay (28 dpm d -1 m -2 ;[START_REF] Beck | Radium mass-balance in Jamaica Bay, NY: Evidence for a substantial flux of submarine groundwater[END_REF] but lower than that reported from Long Island Sound ( > 127 dpm m -2 d -1 ; GarciaOrellana et al., 2014) and the Port of Maó (110 dpm m -2 d -1 ;[START_REF] Rodellas | The influence of sediment sources on radium-derived estimates of submarine groundwater discharge[END_REF].

Figure 5 :

 5 Figure 5: Diffusive 223 Ra and 224 Ra activities as a function of time (not corrected for surface area of core, 0.091 m 2 ).

  bedrock surface of the Öregrundsgrepen seabed and the Börstilåsen esker described as potential discharge zones (Section 2) are prevalent through the shallow bays Tixelfjärden and Kalligafjärden and up to 6000 m off the Forsmark coast (Figure 1.a).Higher average 223 Ra and 224 Ra xs activities were observed around the northern-most transect (t1-5), where the frequency of fractures intersecting the seabed is greatest. Although strong gradients in 226 Ra were not observed here, slight increases in offshore224 Ra xs and 226 Ra activities are associated with the occurrence of these fractures ~1000-1500 and 4000-5000m offshore (3.a, 3.c). Systematic increases in Ra are associated with the fracture zones in each offshore transect, and thus may reflect Ra inputs from these zones.5.3.2 Tixelfjärden bayMaximum brackish water Ra activities were observed in Tixelfjärden bay. Though the influence of 223 Ra and 224 Ra diffusion from sediments is more conspicuous in shallow bays due the larger sediment surface area to water volume ratio,226 Ra and 228 Ra activities, which are not supplied through sediment diffusion, were elevated in this bay by a factor of two relative to the offshore transects. Given high fracture frequency within the bay and the proximity of the Börstilåsen esker alongside the Ca/Mg ratio (Section 5.3.1), SGD is a plausible local source of Ra enrichment. The rate of evaporation in this bay is ~15% the rate of water inflow from the strait (Section 4.3) and cannot explain the Ra enrichment.

Figure 7a- d :

 d Figure 7a-d: Mixing plots of (a) 228 Ra/ 226 Ra ARs, as well as 226 Ra as a function (b) of Ca/Mg ratio, (c) δ 2 H (c), and (d) salinity for deep

Figure 8 .

 8 Figure 8.a-c: (a)δ 2 H and δ 18 O, (b) 223 Ra, 224 R, and (c) 226 Ra as a function of salinity in Kallrigafjärden bay. Stations KF2 and KF3, which deviate from mixing trend near Börstilåsen, esker circled.

J

  sea , the flux of Ra exported to the open sea, is determined from the excess inventory, i.e. the difference between the total 224 Ra xs inventory within the site and that supplied by the open sea (conservatively estimated to 1.3 dpm 100L -1 , Section 5.4.1), divided by the residence time (11 ± 2 d). This results in a J sea of (260 ± 160) • 10 6 dpm 224 Ra xs d -1 . The flux of 224 Ra xs removal through radioactive decay, J λ , is obtained by multiplying the total 224 Ra xs inventory (4800 ± 1700) • 10 6 dpm) by the decay constant of 224 Ra (0.189 d -1 ). The resulting flux of 224 Ra xs removal by decay is (900 ± 310) • 10 6 dpm d -1 .

Table 1 :

 1 Summary of results for the sampled stations. Depth of water column (DPT), distance 198 from shore (m), and salinity alongside Ra activities in dpm 100L -1 , Ba in µg/L, Na and Ca in 199 mg/L. Uncertainties for δ 2 H and δ 18 O were ± 0.6 and ± 0.1, respectively.

	4 Results
	200
	ST	DPT (m)	Sal	Dist (m)	223 Ra	σ	224 Raxs	σ	226 Ra	σ	228 Ra	σ	δ 2 H	δ 18 O	Ba	Na	Ca	Ca / Mg
	t1*	6.2 4.75	220	0.67 0.10	8.1	0.9	12.8	0.6	16.1	1.1 -60.1	-7.9 17.0 1290		0.37
	t2*	13.4 4.71	1450	0.29 0.06	2.0	0.6	12.0	0.6	15.4	0.9 -60.3	-8.0 16.6 1450		0.39
	t3*	6.1 4.72	2730	0.22 0.05	3.7	0.5	13.2	0.7	15.9	1.1 -60.9	-7.9 14.8 1490		0.39
	t4*	11.8 4.63	5490	0.39 0.07	3.0	0.4	15.0	0.6	15.8	0.9 -60.9	-8.0 15.8 1460		0.39
	t5	33 4.64	7810	0.21 0.05	1.5	0.4	14.5	0.6	15.9	1.0 -60.6	-8.0 15.8 1430		0.39
	t6*	10.5 4.82	310	0.30 0.07	2.3	0.7	11.6	1.0	15.8	1.0 -60.5	-8.0 16.9	937		0.36
	t7*	10.6 4.76	1020	0.37 0.06	2.8	0.6	14.0	1.1	15.7	1.1 -60.3	-7.9 16.5 1405		0.39
	t8	13.5 4.71	3100	0.38 0.06	1.7	0.5	13.3	1.6	15.2	3.4 -60.4	-8.0 16.1 1420		0.39
	10 m		4.79		0.21 0.03	1.8	0.5					-60.2	-7.9 16.9 1480
	t9*	16.3 4.70	4040	0.21 0.05	2.1	0.4	15.2	0.4	16.6	2.6 -60.2	-7.9 16.8 1430		0.39
	t10	13 4.72	6130	0.24 0.06	1.4	0.4	13.5	1.2	15.7	2.2 -59.9	-7.7 20.3			0.05
	t11*	8.7 4.74	255	0.38 0.06	2.3	0.5	13.0	1.6	16.8	3.5 -60.4	-8.0 16.9 1250		0.37
	t12*	7.89 4.71	1280	0.20 0.05	1.8	0.4	17.6	1.1	16.1	1.0 -60.0	-7.9 17.0 1010		0.36
	t13	18.5 4.70	2170	0.28 0.06	1.3	0.4	13.8	1.1	15.4	2.4 -60.9	-7.9 18.6 1390		0.39
	10 m		4.72		0.12 0.02	1.3	0.5					-60.4	-7.9 16.6 1460
	t14*	21.5 4.72	2810	0.26 0.06	1.6	0.3	10.0	1.0	17.5	2.7 -60.4	-7.9 15.6 1450		0.38
	t15	20.5 4.72	4360	0.19 0.06	1.0	0.3	16.0	1.4	16.9	2.4 -60.3	-7.9 15.3 1440		0.38
	KF1	4 1.71	1590	1.4 0.18	13	1.2	16.4	1.3	22.9	3.6 -64.8	-8.6 17.9 1170		0.47
	KF2	9.4 4.29	3250	1.2 0.15	11	1.0	21.9	1.4	25.1	3.1 -62.0	-8.1 17.6 1340		0.41
	KF3	7.2 4.19	4270	0.80 0.11	6.8	0.9	20.7	1.6	19.6	3.8 -63.2	-8.5 16.3 1110		0.41
	KF4	8.4 4.74	4940	0.41 0.07	2.4	0.6	18.9	1.1	17.7	1.0 -61.8	-8.1 17.4 1390		0.38
	TF1	3.7 4.84		3.7 0.31	29	2.8	23.5	0.7	38.9	1.4 -58.8	-7.6 19.3 1450		0.39
	TF2	1.6 4.83		2.8 0.18	17	1.9	14.6	1.6	30.2	3.7 -59.6	-7.8 16.0 1470		0.39
	S1		4.79				16	2.5	18.1	0.7	28.4	3.8
	S2		4.67				28	3.6	32.2	2.5	48.1	3.5
	S3		4.81				5.0	1.4	15.6	1.6	16.2	1.1
	Shallow groundwater from piezometer (SFM)
	0001		0.69		8.4 1.25	220	26	328	26	290 23.0 -83.6 -11.5 30.6	165		2.8
	0032		0.55		9.2 1.12	85	9	203	9.0	182	9.0 -84.0 -11.7 50.8	28		9.9
	0049		0.21		3.9 0.55	68	5	50.1	2.5	95	5.0 -81.7 -11.1 21.4	18		8.8
	0059		1.27		5.6 0.74	270	20	262 12.0	287 15.0 -80.5 -11.2 72.5	241		3.0
	0060		0.37		4.6 0.72	73	8	177	9.0	143	8.0 -89.8 -12.6 53.9	5		12.4
	Deep groundwater (KFR)
	07A				288	11	719 240	1670	37 2170 54.0 -70.3	-9.0 32.5 1530		3.8
	01				359	19	2600 290	3100	70 7200	197 -78.7 -10.3 50.6 1050		4.8

194

The measured salinities, Ra isotopes activities, δ 2 H and δ 18 O values, Ba and Ca 195 concentrations and Ca/Mg ratios are shown alongside water column depth and distance from 196 shore for each station in Table

1

.

197

  Ra, 224 Ra xs , 226 Ra, and 228 Ra activities ranged 0.7400 dpm 100L -1 for 223 Ra, 224 Ra xs , 226 Ra, and 228 Ra, respectively. Minimum activities for the four Ra isotopes were found in SFM0049, while maximum activities in SFM0032 ( 223 Ra), SFM0059 ( 224 Ra xs ), and SFM0001 ( 226 Ra and 228 Ra).

4 -1.6, 2 -14, 16 -19, and 18 -23 dpm 100L -1 , respectively. Activities were higher in Tixelfjärden bay: 2.7 -3.7, 17 -29, 15 -24, and 30 -39 dpm 100L -1 for 223 Ra,

224

Ra xs , 226 Ra and 228 Ra, respectively.

In shallow groundwaters, long-lived Ra activities were ~2 to 27-times greater and short-lived Ra activities 0.8 -270-times greater than the brackish water.

223

Ra,

224

Ra xs ,

226

Ra, and 228 Ra ranged 3.3 -10, 63 -290, 48 -350, and 90 -310 dpm 100L -1 , respectively, in shallow groundwaters. Deep groundwaters were more enriched, with activities 2 -4 orders of magnitude greater than the brackish water. Deep groundwaters ranged

280 -380, 470 -2900, 1600 -3200, and 2100 -

  228 Ra/226 Ra ARs are inadequate to define the source of groundwater discharge. Salinity, δ 2 H, and Ca/Mg variations between groundwater and brackish water (Figure7.b-d) are more significant than 228 Ra/ 226 Ra ARs. No clear mixing relation between groundwater and brackish Baltic water for δ 2 H and 226 Ra is observed. However, the Ca/Mg ratios and salinity versus

226

Ra may indicate mixing between groundwater and brackish Baltic water. Provided the elevated Ca/Mg ratio and salinity in Tixelfjärden bay, local discharge of deep groundwater is viable.

  Ra xs activity in the open Baltic Sea is found using the lowest 224 Ra xs activity measured in the studied area (station t15, 1.0 ± 0.3 dpm 224 Ra xs 100L -1 ). The area affected by SGD is then considered to include only those stations with 224 Ra xs activities statistically higher than the open sea 224 Ra xs activity (stations t1-4, t6-7, t9, t11-12, and t14; denoted by * in Table1). Thus, the area influenced by SGD is 16•10 6 m 2 .

	offshore. One such age model, developed by Moore (2006), is expressed through the
	equation:
	τ=	Ra Ra !" 224 228 224 Ra !" ! -Ra 228	224 Ra !" Ra 228 !"# ·λ 224	!"#
	An average depth of 11 m, which represents the depth of the water column averaged over the
	stations constrained as influenced by SGD, is used to constrain the volume of brackish water
	influenced by SGD to 180 • 10 6 m 3 .
	5.4.2 Residence time
	A residence time, which represents the time elapsed since Ra isotopes were transferred from
	their source to the constrained study site, is required to estimate the flux of Ra exported

Table 2 :

 2 Calculated mass balance inputs* and SGD rate ranges.

		224 Ra xs,offshore	Total Inventory			224 Ra xs fluxes (•10 6 dpm d -1 )
		224 Ra xs,obs σ 224Raxs (•10 9 dpm)	J sed	σ sed	J sea	σ sea	J λ	σ λ	J SGD	σ SGD
		2.8	1.7	4.8	1.7	520	31	270	160	900	310	640	350
							SGD rates (m 3 d -1 )		Uncertainty
	SFM0049	68	5					9.5E+05				5.2E+05
	SFM0059	270	20					2.4E+05				1.3E+05
	KFR01	2610	290					2.5E+04				1.4E+04
	KFR07A	11800	830					5.5E+03				3.0E+03
	*Surface area of 16 • 10 6 , an average depth of 11 m, and a volume of 180 • 10 6 m 3 used to calculate
	fluxes.

  , which is two orders of magnitude lower than the224 Ra xs -derived discharge rate of shallow groundwater.Therefore, the SGD rate derived from 224 Ra indicates that a component of recirculated seawater, independent of inland hydraulic head, is important in Öregrundsgrepen. When seawater is driven into the coastal aquifer and discharged back into the sea, the generally short residence time in the coastal aquifer allows the water to become enriched in 223 Ra and 224 Ra, which are rapidly regenerated by aquifer solids, over 226 Ra and 228 Ra[START_REF] Leuk | Radium budget of the York River estuary (VA, USA) dominated by submarine groundwater discharge with a seasonally variable groundwater endmember[END_REF]. Therefore,223 Ra and 224 Ra enrichment has been observed in the absence of 226 Ra and 228 Ra gradients due to the dominance of a short-scale, recirculated seawater component of SGD in Öregrundsgrepen.

Acknowledgements

The authors are grateful to Cecilia Berg, Micke Borgiel, Ignasi Puigdomenech, and Karin Wallner for their assistance in field and laboratory work. This article benefitted from critical reviews by Patrik Vidstrand, Emma Johansson, and Lena Evins. This study was supported by the Swedish Nuclear Fuel and Waste Management Company and the European Union's Seventh Framework Programme for research, technological development, and demonstration under Grant Angreement No. 290336. V.R. has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 748896. Support provided by the Generalitat de Catalunya to MERS (2014 SGD -1356) is also acknowledged.


Dr Alessandro Mattina
email: alessandromattina@gmail.com

David Rosenbaum

Randa Bittar

Dominique Bonnefont-Rousselot

Davide Noto

Maurizio Averna

Eric Bruckert

Philippe Giral

Dominique Bonnefont- Rousselot

Lipoprotein-associated phospholipase A activity is increased in patients with definite familial hypercholesterolemia compared with other forms of hypercholesterolemia

Keywords: lipoprotein-associated phospholipase A2, familial hypercholesterolemia, dyslipidemia, cardiovascular disease, plaque vulnerability, vascular inflammation, cardiovascular risk, statin treatment, high density lipoprotein, low density lipoprotein

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Lipoprotein-associated phospholipase A2

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a calcium-independent enzyme mainly produced by macrophages. Lp-PLA2 has been shown to play a key role in the development of atherosclerosis due to its pro-inflammatory and pro-oxidative effects [START_REF] Bonnefont-Rousselot | Lp-PLA2, a biomarker of vascular inflammation and vulnerability of atherosclerosis plaques[END_REF] . Lp-PLA2 is synthesized within the atherosclerotic plaque and circulates bound to LDL-C particles [START_REF] Davis | Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein[END_REF] . By the hydrolysis of oxidized LDL-C particles, Lp-PLA2 generates bioactive lipid products (lysophosphatidylcholine and oxidized free fatty acids) which can activate and sustain the atherosclerosis process [START_REF] Tew | Properties, Sequencing, and Cloning of a Lipoprotein-Associated, Serine-Dependent Phospholipase Involved in the Oxidative Modification of Low-Density Lipoproteins[END_REF][START_REF] Caslake | Lipoproteinassociated phospholipase A2, platelet-activating factor acetylhydrolase: A potential new risk factor for coronary artery disease[END_REF] . Lp-PLA2 is also implicated in endothelial dysfunction [START_REF] Yang | Lipoprotein-associated phospholipase A2 is an independent marker for coronary endothelial dysfunction in humans[END_REF] , plaque vulnerability [START_REF] Kolodgie | Lipoprotein-associated phospholipase A2 protein expression in the natural progression of human coronary atherosclerosis[END_REF][START_REF] Cola | From Carotid plaque biology to serologic markers of vulnerability to predict the risk of cerebrovascular events[END_REF][START_REF] Weintraub | Identifying the Vulnerable Patient with Rupture-Prone Plaque[END_REF] and has been associated with inflammation within the atherosclerotic plaque [START_REF] Gonçalves | Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation[END_REF] .

Furthermore, elevated Lp-PLA2 levels were related to increased risk of coronary disease, stroke, and vascular mortality in several large scale prospective studies [START_REF] Collaboration | Lipoprotein-associated phospholipase A 2 and risk of coronary disease, stroke, and mortality: collaborative analysis of 32 prospective studies[END_REF] .

However, little data exists in the specific population of patients with familial hypercholesterolemia (FH) which is an autosomal dominant lipoprotein metabolism disorder characterized by elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) [START_REF] Hovingh | Diagnosis and treatment of familial hypercholesterolaemia[END_REF] . FH results from genetic heterozygous (heFH) or homozygous (hoFH) mutation in the low density lipoprotein receptor gene (LDLR), apolipoprotein B-100 gene (APOB) or proprotein convertase subtilisin/kexin type 9 gene (PCSK9) [START_REF] Hovingh | Diagnosis and treatment of familial hypercholesterolaemia[END_REF] . FH patients exhibit early atherosclerotic lesions [START_REF] Parrinello | Carotid atherosclerosis in hypercholesterolemic patients: relationship with cardiovascular events[END_REF][START_REF] Tanner | Endothelium-derived nitric oxide, endothelin, and platelet vessel wall interaction: alterations in hypercholesterolemia and atherosclerosis[END_REF] with premature CVD compared to non-FH patients [START_REF] Descamps | Impact of genetic defects on atherosclerosis in patients suspected of familial hypercholesterolaemia[END_REF][START_REF] Austin | Familial hypercholesterolemia and coronary heart disease: A HuGE association review[END_REF] . Prevalence of CVD among heFH has been estimated to be about 33% [START_REF] Benn | Familial hypercholesterolemia in the Danish general population: Prevalence, coronary artery disease, and cholesterol-lowering medication[END_REF] with a CVD increased risk by 8 to 17-fold vs. non-FH subjects. In patients untreated with statins, angina and acute myocardial infarctions can be observed early in life, even before 40 years old [START_REF] Broome | Risk of fatal coronary heart disease in familial hypercholesterolaemia[END_REF] .

Our aim was to evaluate Lp-PLA2 activity in a population of hypercholesterolemic patients with and without a clinical confirmed definite FH.

MATERIAL AND METHODS

Study population

Patients were consecutively recruited at our outpatient clinic (Cardiovascular Prevention Unit, Institute of Cardiometabolism and Nutrition, La Pitié-Salpêtrière-Charles Foix University Hospital, APHP, Paris, France) between June 2014 and June 2015. Only subjects with hypercholesterolemia were eligible for enrolment in this study. Hypercholesterolemia was defined according to patient's records as: LDL-C >4.14 mmol/L or/and treatment with statin.

FH groups and Dutch Lipid Clinic Score were determined using patient's files. Patients were included and divided into two subgroups:

-Definite FH (DFH) group defined according to the Dutch Lipid Clinic Network criteria [START_REF] Organization | Familial Hypercholesterolaemia (FM) -Report of a Second WHO Consultation[END_REF][START_REF] Nordestgaard | Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: Guidance for clinicians to prevent coronary heart disease[END_REF] All patients underwent routine clinical examination and biological assessments. In addition, Lp-PLA2 activity was assessed. Arterial hypertension (AH) was defined as arterial blood pressure (BP) >140/90 mmHg and/or antihypertensive treatment; type 2 diabetes (T2D) as fasting plasma glucose >6.94 mmol/L and/or glucose lowering treatment. Cigarette smoking (yes/no), body mass index (BMI) (weight in kilograms divided by the square of height in meters) and waist circumference were also evaluated. The study was carried out according to the principles outlined in the Declaration of Helsinki. Approval of the local Ethics Committee was obtained and informed consent was signed by all participants.

Laboratory methods

Lipid analysis

Fresh venous blood was collected in gel-containing Vacutainer® tubes (Becton-Dickinson, Plymouth, UK), then centrifuged at 4,500 rpm at 4°C for 10 minutes.

Fresh serum lipids were analysed by using routine methods on a Konelab 30i analyser (Thermo Electron Corporation). TG were measured by using an enzymatic method with colorimetric detection (Diasys France Condom) [START_REF] Fossati | Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide[END_REF] . Total cholesterol (TC), direct LDL-C and direct HDL-C were determined using automated enzymatic methods (Konelab Thermo Fisher Scientifics, Asnières sur Seine, France) [START_REF] Allain | Enzymatic Determination of Total Serum Cholesterol[END_REF][START_REF] Egloff | Multicenter evaluation on different analyzers of three methods for direct HDL-cholesterol assay[END_REF][START_REF] Bayer | Multicenter evaluation of four homogenous LDL-cholesterol assays[END_REF] . LDL-C was calculated by the Friedewald formula when TG <3.9 mmol/L 24 and directly dosed when TG were between 3.9 and 11.4 mmol/L. ApoA-I, apoB and Lp(a) were measured by immunonephelometry using anti-ApoA-I, anti-ApoB and anti-apo(a) antisera and a BN II nephelometer analyser from Siemens (Siemens Healthcare Diagnostics S.A.S, Saint Denis France; inter-and intra-assay coefficients of variation <4%) [START_REF] Albers | International federation of clinical chemistry standardization project for measurements of apolipoproteins A-I and B. II. Evaluation and selection of candidate reference materials[END_REF] .

Measurement of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity

Measurement of Lp-PLA2 activity was performed with automated colorimetric method on Konelab 30i analyser, by using a rate reaction assay with 1-myristoyl-2-(4nitrophenylsuccinyl) phosphatidylcholine as substrate (Diadexus Inc. South San Francisco.

CA. Eurobio, France). Units were expressed as nmol platelet activating factor (PAF) hydrolysed per minute per mL of serum (nmol/min/mL) [START_REF] Caslake | Lipoprotein-associated phospholipase A2, inflammatory biomarkers, and risk of cardiovascular disease in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER)[END_REF] . All assays were performed in the Department of Metabolic Biochemistry of Pitié-Salpêtrière -Charles Foix Hospital.

Statistical analysis

All continuous variables were described by their mean ± standard deviation (SD) and proportions (%) for categorical variables. The differences between groups were evaluated by Student's t test for continuous variables and χ 2 test for categorical variables. Correlations between Lp-PLA2 and lipid parameters were assessed using Pearson's correlation coefficient except for Lp(a) and TG where Spearman's rank correlation coefficients were provided.

Multivariate linear regression analysis was used to assess the independent contribution of the variables. A p value <0.05 was considered significant. JMP® Statistical Software, Version 11, SAS Institute Inc., Cary, NC, was used.

RESULTS

Our population consisted of 469 subjects. Among them, 118 (25.1%) were classified into the definite FH group. Among DFH subjects, 51 had a known heterozygous mutation of LDLR, APOB or PCSK9 gene. The clinical and biochemical characteristics of the whole population, as well as in DFH and in NDFH subjects are shown in Table 1. The mean Lp-PLA2 activity of the whole population was 187.3 ± 51.2 nmol/min/mL, with a significant difference between males and females (195.9 ± 48.5 vs. 178.0 ± 52.4 nmol/min/mL respectively, p 0.0001, data not shown). No difference in Lp-PLA2 levels was found in the DFH group among subjects with known mutations and subjects without known mutations (210.1 ± 50.5 and 203.8 ± 57.5 nmol/min/mL respectively, p 0.5330, Additional Figure 1). Lp-PLA2 activity was significantly higher in DFH subjects than it was in NDFH subjects (206.5 ± 54.5 vs 180.8 ± 48.4 nmol/min/mL, p <0.0001). DFH subjects were significantly younger than NDFH and less likely to suffer from diabetes or hypertension. Moreover, DFH subjects also exhibited lower HbA1c, TG, ApoA-1 levels, blood fasting glucose and blood pressure (BP) levels than NDFH. In opposite, they had higher TC, LDL-C and ApoB levels than NDFH subjects. Values of HDL-C and Lp(a) as well as the percentage of patients under statin treatment were identical in both groups. Partial correlation of Lp-PLA2 with ApoB and LDL showed that LDL-C and ApoB remains associated with Lp-PLA2 (r adj ApoB = 0.12, p <0.01; r adj LDL-C = 0.12, p <0.01). Non-HDL-C did not remain correlated with Lp-PLA2 when adjusted with LDL-C (r adj LDL-C = -0.03, p 0.86).

In a multivariate analysis, definite definite FH diagnosis as well as LDL-C, HDL-C and statin treatment remained correlated with Lp-PLA2 independently of systolic BP. Inclusion of ApoB in the model in replacement of LDL-C did not change the outcome of the analysis.

Sex was independently related with Lp-PLA2 (Table 3). Separate multivariate analysis in DFH and NDFH patients showed minor differences in Lp-PLA2 determinants but LDL-C and HDL-C remained significantly associated to Lp-PLA2 (Additional Tables 1 and2).

DISCUSSION

In this study, we found that Lp-PLA2 activity in hypercholesterolemic subjects was independently higher in subjects with a clinically confirmed definite FH than in subjects with a non-definite FH according to Dutch Lipid Clinic Network criteria.

Our results extend the results of Tsimihodimos et al. [START_REF] Tsimihodimos | Altered distribution of platelet-activating factoracetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia[END_REF] who found higher Lp-PLA2 activity in FH patients (carrying hetero and homozygous mutations) compared to non-FH hypercholesterolemic subjects and controls in a smaller population. Moreover, in our study, definite FH status and LDL-C levels both independently correlated to Lp-PLA2 activity.

As observed here as well as in some previous studies [START_REF] Yang | Lipoprotein-associated phospholipase A2 is an independent marker for coronary endothelial dysfunction in humans[END_REF][START_REF] Tsimihodimos | Altered distribution of platelet-activating factoracetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia[END_REF] , Lp-PLA2 activity correlates strongly and positively with LDL-C while negatively with HDL-C. The positive relationship with LDL-C may be explained by the fact that Lp-PLA2 is mainly associated with apoB-containing lipoproteins (primarily with LDL), i.e. 70-75% when expressed in mass, and 90-95% when expressed in activity [START_REF] Gazi | Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma[END_REF][START_REF] Tselepis | PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme[END_REF][START_REF] Donato | Advantages of the lipoprotein-associated phospholipase A2 activity assay[END_REF] . This difference in assay procedure could also explain why we found a negative correlation with HDL-C despite about 30% of Lp-PLA2 being carried by HDL particles [START_REF] Cao | Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry[END_REF] . Several lines of evidence suggest that the Lp-PLA2 roles in atherosclerosis pathophysiology may depend on the type of lipoprotein particles with which it is associated to [START_REF] Tellis | Pathophysiological role and clinical significance of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) bound to LDL and HDL[END_REF] . It seems that only LDL-associated Lp-PLA2 exerts pro-inflammatory activity while HDL-associated Lp-PLA2 seems to have an anti-atherogenic role [START_REF] Marathe | Platelet-activating factor acetylhydrolase, and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoprotein particles[END_REF] . HDL prevents or inhibits the formation of LDL-derived oxidized phospholipids [START_REF] Navab | HDL and the inflammatory response induced by LDLderived oxidized phospholipids[END_REF] by the action of at least four specific enzymes present on its surface, including HDL-associated Lp-PLA2. Since oxidized phospholipids are the target of hydrolysis by Lp-PLA2, its activity can be reduced by increasing HDL concentration.

The inverse relationship between Lp-PLA2 activity and HbA1c was also observed in previous studies [START_REF] Mayer | Unexpected inverse relationship between impaired glucose metabolism and lipoprotein-associated phospholipase A2 activity in patients with stable vascular disease[END_REF][START_REF] Onat | Dual activity of serum lipoprotein-associated phospholipase A(2) yielding positive and inverse associations with cardiometabolic risk[END_REF] . It may be explained by the distribution of Lp-PLA2 among lipoprotein particles.

In diabetic subjects [START_REF] Kujiraoka | Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus[END_REF] in particular, Lp-PLA2 is associated mainly with HDL particles and the Lp-PLA2 glycosylation process leads to a greater activity of HDL-associated Lp-PLA2 [START_REF] Tselepis | N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL[END_REF] .

While mendelian randomization studies may suggest otherwise [START_REF] Ueshima | Lipoprotein-associated phospholipase A2 is related to risk of subclinical atherosclerosis but is not supported by Mendelian randomization analysis in a general Japanese population[END_REF] , there are some evidence for an active role of Lp-PLA2 in atherosclerosis [START_REF] Garza | Association between lipoprotein-associated phospholipase A 2 and cardiovascular disease: a systematic review[END_REF] . As Lp-PLA2 hydrolyses oxidized phospholipids in LDL-C particles to release oxidized fatty acids at the sn-2 position and lysophosphatidylcholine [START_REF] Gonçalves | Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation[END_REF] its activity can enhance plaque inflammation [START_REF] Gonçalves | Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation[END_REF] . Hence, upregulation of Lp-PLA2 has been demonstrated within the plaque necrotic core and in macrophages prone to rupture plaques [START_REF] Sudhir | Clinical review: Lipoprotein-associated phospholipase A2, a novel inflammatory biomarker and independent risk predictor for cardiovascular disease[END_REF] . Inflamed vulnerable plaques have been associated with a higher prevalence of coronary heart disease [START_REF] Burke | Coronary Risk Factors and Plaque Morphology in Men with Coronary Disease Who Died Suddenly[END_REF][START_REF] Sidhu | The extracranial carotid artery in familial hypercholesterolaemia: relationship of intimal-medial thickness and plaque morphology with plasma lipids and coronary heart disease[END_REF] in the general population and increased circulating Lp-PLA2 levels have been associated with higher plaque vulnerability [START_REF] Weintraub | Identifying the Vulnerable Patient with Rupture-Prone Plaque[END_REF] . Concerning FH, Caballero et al. [START_REF] Caballero | Detection of subclinical atherosclerosis in familial hypercholesterolemia using non-invasive imaging modalities[END_REF] found an increased prevalence of lipid-rich atherosclerotic plaques in FH using magnetic resonance imaging. Moreover, Van Den Oord et al. [START_REF] Van Den Oord | Assessment of subclinical atherosclerosis and intraplaque neovascularization using quantitative contrast-enhanced ultrasound in patients with familial hypercholesterolemia[END_REF] found that FH subjects more frequently exhibited atherosclerotic plaques neovascularization, a sign of plaque vulnerability. Along with the early and lifelong exposure to elevated LDL-C levels, those observations of more vulnerable plaques in FH patients could partially explain why FH patients develop premature symptomatic atherosclerosis. Our study suggests that the increased CVD risk in patients with FH could be partly explained by the arterial wall inflammation associated with plaque vulnerability and witnessed by high circulating Lp-PLA2 levels. This hypothesis may be hampered by the STABILITY trial [START_REF] Wallentin | Lipoprotein-Associated Phospholipase A2 Activity Is a Marker of Risk But Not a Useful Target for Treatment in Patients With Stable Coronary Heart Disease[END_REF] failure to prove that pharmacological Lp-PLA2 activity lowering could decrease cardiovascular events in a high risk population of patients. However, no specific data on FH patients within the STABILITY trial were published. Also, in another trial [START_REF] Serruys | Effects of the direct lipoprotein-associated phospholipase A(2) inhibitor darapladib on human coronary atherosclerotic plaque[END_REF] , while the darapladib molecule inhibited necrotic core growth of human coronary plaques, it failed to prevent cardiovascular events in a phase III trial.

In our study, statin treatment was negatively correlated to Lp-PLA2 levels independently of LDL-C and definite FH status. This is coherent with the well-known effect of statins which decrease Lp-PLA2 in general populations [START_REF] Braun | Lp-PLA2: A New Target for Statin Therapy[END_REF] but also in selected FH population [START_REF] Ryu | Lipoprotein-associated phospholipase A2 mass and activity in children with heterozygous familial hypercholesterolemia and unaffected siblings: Effect of pravastatin[END_REF][START_REF] Saougos | Differential effect of hypolipidemic drugs on lipoproteinassociated phospholipase A2[END_REF] Our study extend those results in a larger population of real life definite FH patients. Beyond their LDL-C lowering effect, statins exhibit pleiotropic effects. One of them is to stabilize atherosclerotic plaques by reducing foam cell formation and inflammatory process and by inhibiting the macrophages matrix metalloproteinases production [START_REF] Davignon | Beneficial cardiovascular pleiotropic effects of statins[END_REF] . High Lp-PLA2 levels observed in our population may also be explained by the fact that only three quarters of definite FH subjects were under statin treatment. Our results further reinforce the concept that cardiovascular protection with statins may be partially explained by anti-inflammatory effects, perhaps on Lp-PLA2 especially.

Our study exhibits some limitations. First, it is a cross sectional study and therefore we cannot determine causality. Then, we did not analyse the different lipoproteins particles in our study.

Also, we did not measure Lp-PLA2 mass, but Lp-PLA2 activity has several technical advantages [START_REF] Donato | Advantages of the lipoprotein-associated phospholipase A2 activity assay[END_REF] including in the pre-analytic phase that makes it more easy to use in clinical practice. Another limit is that without the certainty of genetic diagnosis, it can be assumed that a variable percentage of FH patients were attributed to the NDFH group. This however did not prevent us from finding statistical differences between the groups.

CONCLUSION

In a population of hypercholesterolemic patients, we found that Lp-PLA2 activity was higher in patients with a clinically confirmed definite FH independently of LDL-C levels and statin treatment in comparison with subjects with a non-definite FH according to Dutch Lipid Clinic Network criteria. These results highlight the particular phenotype of FH within the vast population of patients with hypercholesterolemia. In addition to early and lifelong elevated LDL-C, the high levels of arterial inflammation may also be involved in the premature onset of CVD in FH patients. Their high cardiovascular risk can be prevented by adequate therapeutic strategies using statin anti-inflammatory pleiotropic effects. Our results also reinforce the need for proper identification of this disease which affects 1/200 people in Western countries.

  with a score ≥8; -Non-definite FH (NDFH) group of patients with Dutch Lipid Clinic Network criteria score <8. Exclusion criteria were: LDL-C ≤1.3 mmol/L without lipid lowering treatment, high-density lipoprotein cholesterol (HDL-C) ≥2.6 mmol/L or ≤ 0.39 mmol/L; triglycerides (TG) ≥11.4 mmol/L; fasting glucose ≥10 mmol/L; history or treatment of thyroid disease; FH homozygous status. The aim for those exclusions was to define a homogenous population of patients with hypercholesterolemia.

Table 1 .

 1 Whole population, definite familial hypercholesterolemia (DFH) and non-definite FH (NDFH) subgroups characteristics.

	Characteristics	All subjects	DFH	NDFH	p a
	n	469	118	351
	Age, years	59 ± 14	50 ± 16	62 ± 11	<0.0001
	Sex, men (%)	243 (52)	60 (50.9)	183 (52.1)	0.8084
	Lp-PLA2 activity, nmol/min/mL	187.3 ± 51.2	206.5 ± 54.5	180.8 ± 48.4	<0.0001
	Cardiovascular risk factors
	Current smoking, n (%)	88 (19)	20 (17.0)	68 (19.4)	0.5096
	Type 2 Diabetes, n (%)	74 (16)	6 (5.1)	68 (19.4)	<0.0001
	HbA1c, %	5.9 ± 0.60	5.68 ± 0.55	5.96 ± 0.60	<0.05
	Arterial Hypertension, n (%)	195 (42)	21 (17.8)	174 (49.7)	<0.0001
	Systolic BP, mmHg	116.7 ± 12.8	112.7 ± 12.2	118 ± 12.8	<0.0001
	Treatment
	Antihypertensive treatment, n (%)	186 (40)	19 (16)	167 (48)	<0.0001
	Statins, n (%)	332 (71.0)	90 (76.3)	242 (69.0)	0.1301
	Ezetimibe, n (%)	75 (16)	31 (26.3)	44 (12.5)	< 0.001
	Lipid profile
	Total Cholesterol, mmol/L	5.89 ± 1.58	6.34 ± 1.96	5.75 ± 1.41	<0.001
	LDL-Cholesterol, mmol/L	3.77 ± 1.5	4.36 ± 1.92	3.60 ± 1.26	<0.0001
	HDL-Cholesterol, mmol/L	1.37 ± 0.44	1.42 ± 0.43	1.37 ± 0.45	0.2830
	Triglycerides, mmol/L	1.65 ± 1.32	1.25 ± 0.75	1.81 ± 1.45	<0.0001
	Apolipoprotein A-1, mg/dL	159.3 ± 26.7	155.1 ± 27.4	160.7 ± 26.3	< 0.05
	Apolipoprotein B, mg/dL	112.4 ± 32.6	121.4 ± 40.9	109.4 ± 28.7	<0.001
	Lp(a), mg/dL	43.2 ± 46.0	40.82 ± 44.0	44.0 ± 46.7	0.5237
	a Student's t test p value: DFH vs. NDFH groups. DFH: definite familial hypercholesterolemia; NDFH: non-
	definite familial hypercholesterolemia; Lp-PLA2: Lipoprotein-associated phospholipase A2. BP: blood pressure;

LDL: low density lipoprotein; HDL: high density lipoprotein.

Table 2 displays

 2 Lp-PLA2 univariate correlations between Lp-PLA2 activity and lipid biomarkers. Lp-PLA2 activity was strongly positively correlated with TC, LDL-C and ApoB and negatively with HDL-C and ApoA-1. No correlation was found with TG and Lp(a).

	In DFH patients on statins, Lp-PLA2 activity levels were different from non-definite FH
	patients on statins (202.3 ± 53.8 vs 168.1 ± 43.6 nmol/min/mL, p <0.0001, Additional Figure
	2).

Table 2 .

 2 Univariate correlations between Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and lipid profile in the whole hypercholesterolemic population.

	Lp-PLA2 activity
	r	p

LDL: low density lipoprotein; HDL: high density lipoprotein; NS: not significant. a by Spearman's rank correlation coefficients.

Table 3 .

 3 Variables independently associated with Lipoprotein-associated phospholipase A2 (Lp-PLA2). Beta coefficient ± standard error is shown.

		Lp-PLA2
	R²	β	p

FH: Familial Hypercholesterolemia; LDL: low density lipoprotein; HDL: high density lipoprotein; BP: blood pressure.

ACKNOWLEDGEMENTS

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

AM: study conception, study realization, statistics and manuscript writing; DR: study conception, study supervision, statistics and manuscript supervision; RB: data collection and analysis, manuscript reviewing and scientific supervision; DBR: manuscript reviewing and scientific supervision; DN: statistics and manuscript supervision; MA: manuscript reviewing; EB: manuscript reviewing; PG: manuscript reviewing and supervision.

All authors have approved the final article.