*Article* **Low-Velocity Impact Response of Auxetic Seamless Knits Combined with Non-Newtonian Fluids**

**Vânia Pais 1,2,\*, Pedro Silva 1,2, João Bessa 1,2, Hernâni Dias 3, Maria Helena Duarte 3, Fernando Cunha 1,2 and Raul Fangueiro 1,2**

**Abstract:** Low-velocity impact can cause serious damage to the person or structure that is hit. The development of barriers that can absorb the energy of the impact and, therefore, protect the other side of the impact is the ideal solution for the pointed situation. Auxetic materials and shear thickening fluids are two types of technologies that have great capabilities to absorb high levels of energy when an impact happens. Accordingly, within this study, the combination of auxetic knits with shear thickening fluids by the pad-dry-cure process was investigated. It was observed that, by applying knits with auxetic patterns produced with denser materials and combined with the shear thickening fluids, high performance in terms of absorbed energy from puncture impact is obtained. The increment rates obtained are higher than 100% when comparing the structures with and without shear thickening fluids.

**Keywords:** auxetic; non-Newtonian fluids; low-velocity impact; personal protection

#### **1. Introduction**

Any structure that suffers an impact can be damaged, reversible, or irreversible, depending on factors such as the geometry, the mass, and the velocity of the striker. The frequency of the impact will also affect the result. Low-velocity impacts usually occur in the range of 1–10 m/s depending on the properties of the projectile and the defensive structure. In addition, the properties of the structure will influence the impact consequences [1,2]. These structures can act as a barrier to protect an object or a person located on the opposite side of the impact and can be applied in areas such as sports, automotive, or personal protection [2–4]. In an ideal situation, this structure should absorb as much energy as possible, so that the user or the object that is being protected feels the smallest impact possible [5].

Auxetic materials present a special behaviour when subjected to mechanical forces. These materials have a negative Poissons' ratio since, when they expand laterally, an expansion occurs also in the opposite direction. On the other side, when they are compressed laterally, compression on the opposite side also occurs [3]. Auxetic materials present exceptional properties such as increased mechanical properties, shear resistance, indentation resistance, surface fitting ability, and increased breaking tenacity and energy absorption [3,5–7]. Due to the mentioned topics, auxetic materials are widely applied in the scope of protection.

Shear thickening fluid (STF) is a non-Newtonian fluid that has low viscosity values when under normal conditions. However, when the shear rate is increased to a critical

**Citation:** Pais, V.; Silva, P.; Bessa, J.; Dias, H.; Duarte, M.H.; Cunha, F.; Fangueiro, R. Low-Velocity Impact Response of Auxetic Seamless Knits Combined with Non-Newtonian Fluids. *Polymers* **2022**, *14*, 2065. https://doi.org/10.3390/ polym14102065

Academic Editor: Rajesh Mishra

Received: 20 April 2022 Accepted: 16 May 2022 Published: 19 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

value, the viscosity also increases at a high rate. At this point, the fluid changes to a solidstate [6,8,9]. Due to this ability, the non-Newtonian fluid can have an important role in impact dynamics [10], since it positively influences the mechanical properties and increases parameters performance such as energy absorption [9].

Due to these special behaviours of auxetic materials and non-Newtonian fluids, the combined application of these two technologies in the protection has much potential. The research presented in this paper aims to investigate the mechanical behaviour of auxetic structures produced in a seamless loom as well as the protective effect of this auxetic structure when combined with non-Newtonian fluids. To develop the auxetic seamless knits, two distinct patterns produced with conditions previously optimized by the group were studied. The amount of non-Newtonian fluid, as well as its application by the paddry-cure process, was also studied to obtain a hybrid system with protective properties. Beyond the potential of the application of these two combined technologies, the auxetic structures produced by seamless technology are also an innovative concept and have the high advantage of their reduced weight and high flexibility—when compared to composite structures. Therefore, auxetic knits as well as their combination with STF were optimized within this study in order to obtain materials with increased mechanical performance.

#### **2. Materials and Methods**

#### *2.1. Materials*

The auxetic knits were produced on a seamless Santoni loom, model Top 2 by Playvest (Braga, Portugal). The knits are composed of polyamide (PA) and elastane. The PA is produced by air-jet spinning. Two different types of PA were studied—A 78/68 × 2 and PA 78/68 × 3 and two different knit stitch tightening were studied for each type of polyamide, with P0 being the least tight and P-15 being the tightest. The two auxetic patterns studied are represented in Figure 1. A total of six different combinations was obtained, as represented in Table 1. The non-Newtonian fluids were acquired at Polyanswer.

**Figure 1.** Technical drawing of pattern L and P (**a**) and pattern L and P on seamless knit (**b**).

**Table 1.** Knits studied in the present study.

#### *2.2. Methods*

The auxetic behaviour was studied by imposing the extension of 4 cm of the knits in wales and course directions, separately, with the measurement of the extension or contraction between two specific points in the opposite direction.

The weight of the samples was calculated by dividing the mass of the sample mat by its effective area. The friction coefficients were measured using a binary sensor. This equipment was developed in the University of Minho [11] present on the Frictorq equipment. During this assay, a constant rotational motion was applied between the sample under study and a counter-fabric under a uniformly distributed contact force. The air permeability was measured using an air permeability tester from TEXTEST instruments (Zurich, Switzerland, model FX 3300 and a pressure of 200 Pa was applied with the measurement of the airflow that can pass through the structure.

The tensile, tear, and punction tests were performed by using a dynamometer Mesdanlab Twistronic (Hansfield, Salfords, England). On the tensile test, a tension movement was applied under a constant rate and the maximum force and maximum elongation were measured, according to the standard NP EN 13934-2. The tear test was performed according to the standard ASTM D 2261–2017 and the average of the five highest strength values was determined. The puncture test measures the maximum force and energy absorbed during puncture with a piercing element according to the standards ISO 13996: 1999 and EN 863. The burst test follows the standard NP EN ISO 13938-1 and the burst resistance is measured under constant pressure.

The STF were impregnated on the auxetic knits by the pad-dry-cure process. In this process, the knit goes through a bath and then through two rollers, controlled by pressure and speed.

#### **3. Results**

#### *3.1. Auxetic Behaviour*

The knits produced on the seamless loom without STF were analyzed in order to see if they have auxetic behaviour. The samples with both patterns in the study produced with PA 78/68 × 2 with P0 knit stitch were evaluated. Accordingly, the measurement of the extension or contraction in the opposite direction in which the extension is executed was performed. The results are represented in Figure 2. Note that the wales and courses mentioned in the legend refer to the opposite direction in which the movement is imposed. The positive values represent the situation where the extension in one direction is followed by the extension in the opposite direction. Accordingly, the positive values represent the samples that have auxetic behaviour. Concerning the knits with the L pattern, it is observed that when the extension is imposed in the course direction, the opposite direction, i.e., the wale direction, always has a positive value. Thus, the L pattern has auxetic behaviour in the direction. In knits with the P pattern, the wales also have higher values; however, the final value of the extension is equal to zero. Thus it can generally be concluded that the L pattern has a greater prevalence of auxetic behaviour than the P pattern. This auxetics nature only happens when the extension happens in the course direction, being the wales direction the side that expands laterally. Hu et al. also concluded that, when the strain happens in

the course direction, a higher auxetic behaviour is observed. The reason pointed out by the authors for this behaviour is the fact that, in the wale direction, the stripes are closer. Accordingly, when the course direction is extended, a higher transversion expansion effect occurs [12].

**Figure 2.** Extension of knits with L and P patterns in wales and course direction when imposed extension in the opposite direction. The *x*-axis represents the strain being applied.

#### *3.2. Structural and Mechanical Characterization*

The auxetic knits without STF were characterized and analysed to study their protective behaviour and their comfort parameters. The knits weight, specific air permeability (SAP), and the friction coefficient values (COF) of the L and P patterns produced in the several conditions in this study are shown in Figure 3. Regarding the weight values, it is observed that the L pattern has smaller values. Accordingly, during the production of the L knit, less raw material is required. It is also observed that when the knits are produced with a denser PA-PA 78/68 × 3-higher weight values are obtained. This result is logical since, in this situation, a higher amount of material is present in the same area. The change in the degree of tightness does not seem to promote changes in weight as significantly as the other parameters since no behavioural trend is observed. These results will directly influence the SAP values since denser structures limit the passage of air with greater impact due to the higher obstruction of pores [13]. The obtained results fall under this statement since the samples with denser PA have smaller SAP values. In COF values it is observed that the L pattern has higher values, regardless of production conditions. When a denser PA is applied it is also observed that the COF values are higher. However, the difference between the several values is small and in some samples is within the range of standard deviation values. Accordingly, it can be concluded that denser PA promotes

the production of a more compact knit, which, in turn, will decrease the amount of air able to pass through the structure and will increase the friction intensity. The weight and air permeability parameters will affect the comfort parameters and they will influence properties like moisture management and thermoregulation [14]. Additionally, the friction will also affect the comfort parameters since when the skin contacts a surface, a touch perception occurs. Moreover, higher COF values may cause damage to the skin [15]. The obtained values fall under the ones referred by other authors [15,16].

**Figure 3.** Weight, SAP and friction coefficient of knit samples.

The mechanical behaviour of the knits was also studied and were applied to different types of tests a tensile test, a tear test and a burst test. For each analysis, the corresponding maximum force achieved was analysed. The obtained results are represented in Figure 4. In the tensile test, regarding the L pattern, the maximum force values achieved are very similar in both knit directions—the wale and the course. On the other hand, in the P pattern differences were observed depending on the direction in which the tension is applied, obtaining higher values in the course direction. In Figure 1 it is possible to see that the L pattern has a very similar structure in both directions. However, the P pattern has different points depending on the observation direction. Due to this difference, the structure will have different behaviours depending on the direction in which the tension is applied. Yet, despite the P pattern having the highest force values in the course direction, it also has the smallest values in the wale direction. Accordingly, the L pattern may have much potential due to its intermediate values, not compromising its effectiveness in the wale direction. Concerning the types of PA, it is observed that the denser PA has the highest values of maximum force. Thus this compact arrangement will positively reinforce the structure, allowing it to achieve better mechanical performance. It is also important to note that the knit with the P pattern has a maximum force value in the course direction

that is much higher than the other samples. This difference must be related to production problems instead of some standout mechanical properties of this sample, since this result is not observed in the other analysis and has no logical reason to happen. Accordingly, this result should be ignored in the analysis of this study. In the tear test, these differences imposed by the different directions were less noted; however, the same conclusions were observed, i.e., higher maximum forces are achieved when the PA 78/68 × 3 is applied. In the burst test, once again, the denser sample has the higher force values. Besides that, it was observed that the L pattern has much higher force values. In this final test, both knit directions are evaluated at the same time when executing one test since the assay is performed using a round sample. Accordingly, the differences previously observed in both directions of the P pattern will be conjugated in the final result. Due to this, the burst results with knits with the P pattern had much smaller force values than knits with the L pattern. Generally, concerning the mechanical parameters of the knits, it can be concluded that denser materials positively impact the achievement of higher force values. Samples with a tighter stitch seem to generally have higher force values, however, with a low impact.

**Figure 4.** Maximum forces obtained in the tensile test, tear test and burst test.

#### *3.3. Combination of Auxetic Knits with STF*

The auxetic knits were combined with the STF to increase their protection capability. Other authors also studied the combination of these fluids with other materials to boost their protective properties. Santos et al. proved that the addition of STF to Kevlar fabrics improves the performance of these substrates under impact tests [17]. Lu et al. and Santos et al. also demonstrated that the combination of STF with materials like wale-knitted spacer fabrics and Kevlar woven samples, respectively, can be applied as a material for personal protection [18,19]. Within the present study, STF were combined with the auxetic knits by the pad-dry-cure process. The impregnation rates changed from 60 to 70%. The knits with L and P patterns, before and after STF impregnation, are shown in Figure 5.

**Figure 5.** Knits with L and P patterns, with and without STF.

Once more, tensile tests were performed to see the mechanical behaviour of the samples. However, in this turn, the absorbed energy was also calculated to see the potential of the obtained materials to be applied in protective applications. The materials' ability to absorb energy is important in protection products due to the increase in the distribution of loads throughout the hierarchical architecture [20]. The graphical results obtained by knits with the L and P patterns are represented in Figures 6 and 7, respectively. When performing the tensile tests, it was observed that the knits impregnated with STF could stretch more. This higher stretch capability boosts the achievement of higher absorbed energy values. It was observed that when knits were combined with STF, higher maximum force and absorbed energy values were obtained. The only exception was the P pattern in the course direction. However, the incongruity of this sample was already mentioned in the analysis of the mechanical behaviour of the knits without STF. When comparing the L and P pattern, it is observed that the P pattern has higher energy absorbed values, making the knits stand out where denser PA was applied. This result follows the ones mentioned before, where it was concluded that applying higher amounts of mass in the same area unit increases the capability of the samples to support higher forces in the tensile movement. Moreover, as a consequence of this property, the samples with denser material will have the ability to absorb higher amounts of energy and much more potential to be applied as a protective material/product. Yan et al. also studied the influence of material density on protective applications' performance. In the mentioned study it was observed that when materials have higher densities they can reach higher forces when burst and puncture tests are performed. The higher amount of fibers and bonding points per unit of the area of materials with higher densities are the reasons given in the study for achieving higher mechanical behaviours [21].

**Figure 6.** Maximum force and absorbed energy of knits with L pattern obtained on tensile tests.

**Figure 7.** Maximum force and absorbed energy of knits with P pattern obtained on tensile tests.

To simulate a real low-velocity impact, puncture tests were performed with the calculation of maximum force and absorbed energy. The obtained results of knits with the L and P patterns—with and without STF—are represented in Figure 8. Once more it was observed that the addition of STF will increase the mechanical and protective behaviour of the samples in this study and that the P patterns have a higher performance in terms of mechanical behaviour. The samples with denser PA and impregnated with STF generally had higher values of energy absorbed, especially in the situation in which the stitch is tighter. These two combinations are the perfect match to correspond to the points mentioned by Yan et al., i.e., higher amount of fibers and bonding points per unit of area [21]. This combination will increase the protective effect of the samples.

**Figure 8.** Maximum force and absorbed energy of knits with L and P pattern obtained on punction tests.

#### **4. Conclusions**

In this research, a combination of auxetic knits with STF was studied. First, the auxetic knits were characterized, and it was concluded that denser materials increase the mechanical performance of the final structure. When both technologies are combined through the pad-dry-cure process, a higher increase in mechanical behaviour happens. Moreover, puncture tests were performed to simulate the low-velocity impact and it was concluded that the absorbed energy increases with the presence of STF, being more visible in the knit with the P pattern. The increase of materials density, as well as the tightness stitch, will increase the protective behaviour mentioned. The product resulting from the combination of the technologies in this study is very light when compared to the most protective products on the market. Accordingly, this is a promising approach to be applied in personal protection applications.

**Author Contributions:** Conceptualization, V.P.; Investigation, V.P., H.D. and M.H.D.; Methodology, V.P., P.S., H.D. and M.H.D.; Project administration, P.S. and J.B.; Resources, H.D. and M.H.D.; Supervision, F.C. and R.F.; Writing—original draft, V.P.; Writing—review & editing, V.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Compete 2020 grant number 45211.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*3.7. Loss of Hypermucoviscous Phenotype with Colistin Selection*

Our results indicated that rapid COL-R was initially dependent on mutations in mucoid phenotype A regulator gene *rmpA*, leading to changes in capsular polysaccharide synthesis and the hypermucoviscous (HMV) phenotype, independent of bacterial lifestyle. To demonstrate the *rmpA* mutations and alterations in HMV, we conducted the string test for hypervirulence on 1 day evolved (under 1 2 MIC colistin treatment) planktonic population clones compared to the evolutionary ancestor clone. We found a significant decrease in the percentage of colonies that passed string tests following 1 day of colistin treatment for all three planktonic populations (Supplementary Figure S2). These results suggest that there may be an evolutionary tradeoff between hypervirulence and progressive resistance to colistin.

### **4. Discussion**

Colistin resistance has been extensively studied with meaningful mutations and their roles in the resistance mechanism identified. Modifications in lipid A moiety of LPS, overexpression of two-component regulatory systems PhoPQ and PmrAB, plasmid-mediated transfer of mobilized colistin resistance genes *mcr-1* to *mcr-8*, and the inactivation of the PhoQ/PhoP signaling regulator MgrB are some of the most understood mechanisms of colistin resistance in Gram-negative bacteria [14,37,44–50]. In this study, we narrow the knowledge gap in understanding the importance of mutation timing in the progression of COL-R by gene function for both planktonic and biofilm KP lifestyles. We show a similar pattern of resistance mutation timing through colistin selection between bacteria lifestyles (Figure 4). The "first wave" of early resistance was consistent between lifestyles, with the generation of mutations relevant to capsule production, cell membrane integrity, and energy metabolism. Mutations in *mscL* and *nadA*, regulating osmotic stress and energy metabolism, respectively, were likely immediate responses to environmental stress posed by colistin treatment. NadA is a catalyst in the biosynthesis of nicotinamide adenine

dinucleotide (NAD+), an essential cofactor, signaling molecule, and coenzyme for redox reactions of energy metabolism [51,52]. MscL is a pore-forming membrane protein that protects the cell from osmotic downshock, by promoting the efflux of various molecules such as potassium, glutamate, and proline, from the cytosol [53]. MscL is considered a potential drug target by acting as an emergency release valve, allowing for the uptake of extracellular molecules, including antibiotics [54]. These mutations may promote the development of further resistance mutations with roles in LPS modification and synthesis.

Following the "first wave", sporadic mutations affecting LPS structure were observed after day 2 in planktonic populations and occurred later on, by day 6, in biofilm populations. It appears that the positioning of the two-component system sensor and response regulator mutations responsible for LPS modifications may impact COL-R progression. For instance, for planktonic and biofilm populations, mutations at unique gene positions were generated in *phoQ* and *pmrB*. It is possible that mutation positions facilitating optimized COL-R became fixed in each population through enhanced selection pressure. Interestingly, mutations in LPS biosynthesis, likely leading to LPS loss, were generated following modifications in lipid A. For both lifestyles, lipid A modifications preceded mutations in *lpxC* and *lpxD* genes responsible for LPS biosynthesis. This suggests that LPS production may have been altered through colistin selection. Complete loss of LPS is a known mechanism of COL-R in *A. baumannii*, through modifications in *lpxA*, *lpxC*, and *lpxD*, the primary genes involved in lipid A synthesis, leading to a dramatic increase in colistin MIC of greater than 256 μg/mL [49]. Loss of LPS through deletions in *lpx* genes associated with COL-R has also been observed in *Escherichia coli* [55]. However, less is understood regarding the potential loss of LPS in KP, leading to rapid COL-R. Decreased LPS and modifications in LPS are likely the dominant mechanisms of COL-R observed in both planktonic and biofilm evolutions in our study. In addition, an uncharacterized gene mutation (*IT767\_0158*) was detected in both lifestyles following mutations *lpxC* and/or *lpxD*, suggesting that this gene may have roles in regulating LPS. Further investigation is necessary to understand the role of these gene mutations and whether their function is related to LPS biosynthesis.

We also found that bacterial evolution to resistance is likely specialized according to bacterial lifestyle in terms of mutation timing and MIC increase. The biofilm environment itself is designed to protect and respond in defense to environmental stressors, shown by a lag time in COL-R development compared with planktonic-evolved populations. In addition, biofilm development requires more time and metabolic demand for cell attachment, colonization, and maturation, compared to free-living cell propagation. Several mutations were theoretically specialized for biofilm-evolved COL-R, including mutations with roles in peptide transport, fatty-acid biosynthesis, and biofilm formation (*sbmA*, *fadR*, *acpP*, and *qseC*). The sensory kinase QseC is part of the two-component-based quorum sensing system (QseBC), which responds to environmental stress, including changes in osmotic pressure, heat shock, and oxidative stress [56]. Further investigation is required to understand the role of the quorum sensing system QseBC in COL-R development. Mutations generated at later timepoints (on or past day 27) had roles in regulating LPS modifications (*mgrB*, *phoP*, and *arnC*), as well as the envelope stress response and efflux pump expression (*baeS*). Mutations in *baeS*, generated by day 36 for both lifestyles, were likely attributed to bacterial defense against interactions of colistin at the bacterial cell membrane. The membrane-bound sensor histidine kinase BaeS is part of a two-component system involved in envelope stress response that responds to environmental stressors and regulates the expression of different efflux pumps [57]. The pattern of COL-R mutation timing between bacteria lifestyles appears to be both similar and specialized due to growing metabolic demands and likely differences in bacteria susceptibility to colistin treatment.

Through our long-term 36 day experimental evolution, we were able to monitor changes in mutation position and frequency and addition of newly acquired mutations, with the intent to better understand how KP continues to adapt to increasing colistin pressure. These methods allowed us to assess the temporal regulation and interactions of COL-R mutations for over 374 generations (36 days × ~10.2 generations per day). Additionally, we were able to study the timing of various impacted COL-R genes and their functions by bacterial lifestyle. By performing the experimental evolutions using both planktonic and biofilm growth, we were able to account for the effects and demands of bacteria lifestyle as a part of the COL-R mechanism and compare/contrast the most beneficial mutations that led to substantial colistin MIC increase with enhanced selection pressure.

A limitation of in vitro experimental evolution studies is the inability to capture the resistance mechanisms associated with plasmid-mediated transfer in addition to somatic gene mutations. As expected, we did not observe any horizontal transfer of plasmid-borne COL-R genes such as the *mcr* variants, which have been rising in prevalence in nature and human patients [15]. Nonetheless, the system of in vitro experimental evolutions under a controlled environment allows us to unmistakably identify the most beneficial chromosomal alterations leading to progressively enhanced colistin resistance.

Considering the rapid resistance to colistin detected for KP in both lifestyles, it is crucial to determine if the COL-R isolates that could exist in clinical settings will remain susceptible to the newly approved β-lactam/β-lactamase dual-inhibitor antibiotics. CAZ/AVI has been approved for administration in the United States since 2015 to combat ESBL and carbapenemase-producing infections. Avibactam prevents ceftazidime hydrolysis by carbapenemases (KPCs) and ESBLs, while the third-generation cephalosporin ceftazidime exhibits bactericidal activity by inhibiting cell-wall synthesis [58]. Our results demonstrate the diversity in bacterial resistance mechanisms to various antimicrobials, including AMPs and conventional dual-inhibitor antibiotic treatments designed to prevent resistance development. These results support that CAZ/AVI remains a critical treatment option for colistin-resistant infections, which is increasingly vital with the rise in high mortality and hypervirulent MDR bacterial infections in the clinical setting.

In summary, this study presents a clearer understanding of the timing and significance of COL-R mutations in KP to ultimately aid in the development of new clinical treatments that successfully eradicate and/or prevent CR-KP infections in patients. Experimental approaches using in vitro and in vivo models of antibiotic selection similar to laboratory systems described here and isolates sampled from patients before and after treatment [59,60] would facilitate the identification of potentially critical resistance mutations. In addition, we can study the influence of epigenetic factors such as DNA methylation of bacteria, which regulate the expression of genes [61]. With further gain- or loss-of-function studies to investigate the impact of individual and combinations of mutations on antimicrobial resistance [62–64], these data are useful for the clinical situation by elucidating the critical targets that are utilized by bacteria for adaptive resistance to antimicrobials. Along with understanding the influence of timing of critical mutations and timing of affected genes by their functional roles for cell survival through adaptive evolution to resistance, studies like this will be important in enhancing drug development to prevent or lessen the rapid resistance to antimicrobials.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/pharmaceutics15010270/s1: Figure S1. Schematic of planktonic and biofilm experimental evolution workflow; Figure S2. Evolved KP isolates quickly lost their hypervirulence.

**Author Contributions:** Conceptualization, J.M.K. and Y.P.D.; formal analysis, J.M.K. and Y.P.D.; investigation, J.M.K. and Y.P.D.; funding acquisition Y.P.D.; resources, Y.P.D.; supervision, Y.P.D.; writing—original draft, J.M.K.; writing—review and editing, Y.P.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Institutes of Health (R01 AI-133351 to Y.P.D.). The funding agencies had no role in the study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Materials. The sequencing data have been deposited with links to BioProject accession number PRJNA922161 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA922161).

**Acknowledgments:** The authors thank Jaydep Halder and Pranav Kumar Kaliperumal for their assistance in carrying out string test experiments.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.


*Article* **Intake of Sugar-Sweetened Beverages in Adolescents from Troms, Norway—The Tromsø Study:** *Fit Futures*

#### **Guri Skeie 1,\* , Vårin Sandvær 1,2 and Guri Grimnes 3,4**

Received: 1 November 2018; Accepted: 17 January 2019; Published: 22 January 2019

**Abstract:** High intake of sugar-sweetened beverages (SSB) has been associated with weight gain and chronic disease. The objective of this paper was to study the intake of SSB and characteristics associated with SSB intake in adolescents from Troms, Norway. We present results from a cross-sectional analysis from the Tromsø Study: *Fit Futures*, with 426 female and 444 male students aged 15–17 years (93% participation rate). Descriptive statistics and logistic regression analyses were performed. Among females, 31.8% drank at least one glass of SSB per day on average, compared to 61.0% among males. The adjusted OR (odds ratio) of daily SSB drinking for males vs. females was 3.74 (95% CI (confidence interval) 2.68–5.22). Other dietary habits such as eating snacks, drinking artificially sweetened beverages, fruit juice, and seldom eating breakfast were associated with higher odds for daily SSB drinking, as was daily snuffing. Weight class was not associated with daily SSB drinking. Students in vocational studies, particularly males tended to be more likely to be daily SSB drinkers. The prevalence of participants who on average were daily drinkers was higher than in national studies. We have identified several possible targets for interventions. Clustering of unhealthy behaviours and tendencies to socioeconomic differences are of particular concern.

**Keywords:** adolescent; dietary behaviour; nutrition; Norway; sugar-sweetened beverages

#### **1. Introduction**

High intake of sugar-sweetened beverages (SSB) [1] has been associated with several health outcomes. Estimates from the Global Burden of Disease collaboration suggest that worldwide, 184,000 deaths per year are attributable to SSB consumption, mainly due to type 2 diabetes (*n* = 133,000), but also due to cardio-vascular diseases, and cancer [2]. A recent review on diabetes type 2 suggests a 13% higher incidence per serving per day, after adjustment for adiposity [3]. High sugar intake leads to dental decay [4], and SSB is one of the major sugar sources in many demographic groups, including Norwegian youths [5,6]. High SSB intake has also been associated with dental erosion, weight gain, and obesity, although the evidence is not unequivocal [7–9]. While it is the high sugar content that is associated with dental caries, the dental erosion is due to acidity [4,7], and therefore, replacement of SSB with light or artificially sweetened beverages might be beneficial for caries, but not have much effect on erosions. Dental caries has been the most common chronic disease of childhood [10], and damage to the permanent teeth due to caries or erosion, cannot be reverted.

The intake of SSB has been high in Norway, and the authorities have taken initiatives to reduce the intake [11,12]. In the period this study is covering, the aim was to reduce the number of daily consumers of SSB with 20% [13]. Over time, the trend in SSB intake among Norwegian children and adolescents has changed: Nationally representative cross-sectional studies performed in 1989–2001 as part of the international Health Behaviour in Among School-aged Children (HBSC) study, showed both a clear increase in frequency of intake of SSB and an increase in daily users over time [14]. More recently, from the 2005/06 survey, the reported intake has been lower, though not with a continuous decreasing trend [11,15]. In 2014, 11% of the males and 5% of the females drank SSB daily [16]. Another study comparing cross-sectional data from 11–13-year-old children before and after initiatives to reduce SSB consumption showed that the intake of lemonade and regular soft drinks, decreased, the intake of diet soft drinks increased, while juice consumption increased in males, and decreased in females [12].

A higher intake or frequency of intake, of SSB among males [12,15,17,18] and those with lower socio-economic status [12,17] is commonly reported, but in the HBSC study, there was no socio-economic difference in SSB consumption [11]. Some have questioned whether the association between SSB intake and type 2 diabetes is due to the sugar content of the SSB or related lifestyle factors such as other dietary practices, or (lack of) physical activity [19]. Intake of SSB has been associated with lifestyle factors such as physical activity and smoking [18]. Finally, consumption of SSB has been associated with parental modelling and regulation [20–22]. Earlier studies have suggested both poorer dietary habits among adolescents in Northern Norway [23], and found higher prevalence of dental caries as compared to southern parts of the country [24,25]. Our aim was to assess the proportion of daily consumers of SSB in 15–17-year-old adolescents from Troms, Norway, and their characteristics.

#### **2. Material and Methods**

The Tromsø study: *Fit Futures* is a population based longitudinal study with repeated measures of various indicators of lifestyle and health among adolescents [26]. The current paper is a cross-sectional analysis from the first survey in 2010–11, where all 1st grade students from all the eight upper secondary schools in Tromsø and Balsfjord municipalities were invited to participate. The catchment area includes both urban and rural populations. In 2010, 1301 students were enrolled as 1st year upper secondary school students, but 70 persons quit before *Fit Futures* 1 was conducted. Furthermore 114 students were sick or not reached for other reasons, leaving 1117 students who were invited to participate [26]. From those invited, 1038 students attended the study, giving a 93% participation rate.

Students were given time off from school and transported to the Clinical Research Unit of the University Hospital of North Norway, where trained personnel performed anthropometrical measurements, took blood samples, performed physical examinations, and conducted clinical interviews. The students completed self-administered digital questionnaires on a variety of health and lifestyle topics in addition to those detailed below. Information regarding school study programme was collected from school records. The Tromsø study: *Fit Futures* has been described in more detail elsewhere [26].

The age of the participants ranged between 15 and 28. Adolescents following a typical Norwegian educational progress are between the ages of 15–17 at 1st year of upper secondary school, and therefore students 18 years and older were excluded from the analyses (*n* = 77). Exclusions were also made for those missing data on variables used in the main analyses (*n* = 95), which therefore included 870 students, 426 females and 444 males.

The Norwegian Data Protection Authorities and the Regional Committee for Medical and Health Research Ethics have approved the *Fit Futures* study (ref no 2009/1282 and 2012/1904). The study was conducted in accordance with the Declaration of Helsinki. All participants in *Fit Futures* signed an informed consent declaration. For students under the age of 16 additional written consent was provided by their guardians.

#### *2.1. Dietary Variables*

The questionnaire assessed consumption frequency of 14 different foods/food-groups and 10 different beverages. The questions on diet can be found in supplementary file 1. Questions regarding beverages had the response categories "seldom/never", "1–6 glasses per week", "1 glass per day", "2–3 glasses per day" and "4 glasses or more per day". The replies to the two questions on sugar-sweetened carbonated and non-carbonated soft drinks were summed based on the category midpoints (e.g., if they answered 2–3 glasses per day on both questions, it would be counted as 5 glasses per day). A binary variable was constructed, distinguishing between those who, on average, drank SSB daily and not. Similarly, the two questions on light/artificially sweetened carbonated and non-carbonated beverages were combined into one, as were the four questions on milk/yoghurt. The new variables were categorized in order to avoid low cell counts.

For fruits and vegetables (two questions), the categories were "seldom/never", "1–3 times per month", "1–3 times per week", "4–6 times per week", "1–2 times per day", "3–4 times per day" and "5 times per day". These variables were combined into one variable for number of fruits and vegetables eaten per day. This variable was not normally distributed, and therefore recoded into a categorical variable with four categories, based on the distribution. The variables for sweets (e.g., chocolate and drops) and sweet and savoury snacks (e.g., potato crisps, cakes, cookies, buns) had the same categories as the fruit and vegetables, except "every day" was the highest frequency. Again, categories were combined on a-variable-to-variable basis in order to avoid low cell counts.

We assessed frequency of eating breakfast, dinner (the main hot meal), and lunch. As Norwegian students are not served meals at school, those who do not bring their lunch from home (usually sandwiches) often buy something to eat at some nearby store/kiosk, or possibly a canteen. Therefore, bringing lunch from home could be a marker of a healthier diet, or stronger parental control. For breakfast and dinner, the response categories were "every day", 4–6 days a week", "1–3 days a week" and "seldom/never". For lunch brought to school, categories were "every school day", "3–4 days a week" "1–2 days a week", and "seldom/never". Breakfast and lunch were recoded into three categories covering seldom/never, most days, and every (school) day. Dinner was recoded into every day/not every day.

#### *2.2. Other Variables*

Study program was classified as general studies (including sports and physical education studies) and vocational studies. We combined the information about physical activity from two questions: First, the students indicated whether or not they were doing sports or physical activity (such as skateboarding, soccer, dancing or running) outside school hours. Then, weekly sports/physical activity outside school hours was assessed with the original categories: "none", "about 30 min", "about 30–90 min", "about 2–3 h", "about 4–6 h", "7 h or more". Those who on the first question indicated no activity were combined with the lowest group on the second question and coded none. The next three categories for weekly activity outside school hours were coded as up to three h a week, and the last two as 4 h or more.

Students were asked about average time spent in front of computers, TV, DVD or similar outside of school hours, differentiating between school days and weekend days. Categories were the same as for physical activity, except for the highest categories: "about 7–9 h" or "10 h or more". School days and weekend days were weighted 5/7 and 2/7 respectively and combined into one variable. The variable daily screen time was split into categories of "<2 h", "2–3.99 h" and "≥4 h" per day.

Students were asked about smoking and snuff (or snus, a form of smokeless tobacco, a moist powder tobacco placed under the upper lip) habits, with response categories "no, never", "sometimes" or "daily". Due to few daily smokers, they were combined with occasional smokers. Students were asked how often they drank alcohol, with options "never", "once per month or less", "2–4 times per month", "2–3 times per week" or "4 times or more per week". The highest categories had few responders and were combined into the category "2 times per month or more".

A question on who the students lived with (some form of family guardian, friends or alone) was used to differentiate between those who had moved out of home and not. Students answering that they lived both with parents and had moved out of home (*n* = 7) were assumed to be commuters and were therefore included in the group that had moved out of home. Only two students answered living in an institution (and having moved out of home). They were still included in the group living at home, as it is likely that an institution would have an adult in charge of the food environment.

BMI was calculated as weight in kg divided by the square of height in meters (kg/m2) and Cole and Lobstein's revised gender- and age-specific cut-offs [27] were used for classification of BMI categories. Due to few thin and obese students, these groups were merged with the categories normal weight and overweight, respectively.

Students were asked about parents' educational level. The categories "primary school" and "upper secondary school" were combined due to few responders. Furthermore, this variable differentiated between higher education for less and more than four years. The category "do not know" was combined with "missing". Since around 25% of the students did not know or did not answer the questions on mother's and father's education, the variables were only used for additional analyses, and not introduced in the main statistical model.

#### *2.3. Statistical Analyses*

Descriptive and analytic statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Two-sided *p*-values < 0.05 were considered statistically significant. As the variables included in the analyses were categorical, unadjusted analyses were performed using contingency tables with chi-square tests, with the percentage of students that on average reported daily drinking of SSB as outcome. Data from all participants were used to calculate the percentage daily drinkers, but the numbers for non-daily drinkers have been suppressed for increased legibility. Adjusted analyses of characteristics associated with daily drinking of SSB were performed using logistic regression. We considered health related characteristics (smoking, using snuff, drinking alcohol, physical activity, screen time, body mass index), school programme, whether the student lived with parents/guardians, dietary variables (fruits and vegetables, sweets, snacks, beverages other than SSB, and meals eaten) as potential covariates. As there was a large difference in SSB drinking between sexes, both unadjusted and adjusted analyses were performed for each sex separately. However, in order to compare the results obtained between sexes, the adjusted logistic regression model was constructed by including independent variables which were associated with daily drinking of SSB (*p* < 0.25) in the unadjusted analyses of the total sample, weighted for sex. Variables were manually excluded until only significant covariates remained in the model. An additional model was constructed including also sex, mother's and father's education, in order to estimate the effect of sex, and study socio-economic differences from an additional perspective. Father's education was not a significant covariate and was not included in the final analyses. To assess potential sex differences in the associations, we fitted interaction terms with sex, and re-ran the regression analyses for each covariate.

No indication of multicollinearity was observed when evaluating the variance inflation factor. Hosmer and Lemeshow's goodness-of-fit test was used to evaluate overall fit of the regression models. To evaluate how much of the variance in daily drinking of SSB the model explained, pseudo *R*2 (Nagelkerke/Cragg and Uhler's) was assessed.

#### **3. Results**

The mean age of the participants was 16.1 year (Table 1). While most females were in general study programs (61.7%), most males had chosen vocational studies (55.0%). Most students lived with their parents/guardian (females 85.9% and males 87.8%). More males (61.3%) than females (32.2%) reported drinking SSB on average every day (*p* < 0.001). Females tended to drink water more frequently than males, while males tended to drink milk more often than females. About half of the students had breakfast daily, and about one-third brought lunch to school every school day, females slightly more often than males. Almost 60% of the females and 73% of the males had dinner daily.

**Table 1.** Characteristics 1 of the participants in the Tromsø study: *Fit Futures.*

**Table 1.** *Cont.*

1 Mean age, otherwise percentage distributions. 2 Sum of four different milk types (different fat content) and yoghurt. Includes both plain and fermented milk. 3 Weighted mean of weekends and weekdays.

The prevalence of overweight/obesity was high, 19.3% in females and 23.7% in males (Table 1). A larger proportion of males than females was in the group with the highest screen time (47.7% vs. 35.0%). Regarding physical activity outside school hours, the distribution in females and males seemed to be similar. While 20.0% of females and 23.4% of males smoked occasionally or daily, 32.9% of females and 39.6% of males used snuff occasionally or daily. The proportion of students reporting never drinking alcohol was quite low (females 23.5%, males 32.7%).

More than 50% of the females and males drank carbonated SSB 1–6 times a week, but more males than females drank them daily (Figure 1a). Non-carbonated SSB was drunk less frequently than carbonated SSB, and the majority reported drinking them seldom/never, though males also consumed these beverages more often than females (Figure 1b).

**Figure 1.** Intake of (**a**) carbonated sugar-sweetened beverages (SSB); and (**b**) non-carbonated SSB, by sex.

In univariate analyses, living with parents/guardians was not associated with daily SSB drinking, neither in females nor in males (Table 2). Almost 70% of males in vocational studies were daily drinkers, compared to half of the males in general studies. In females the corresponding percentages were slightly less than 50 in vocational studies and just above 20 in general studies (*p* < 0.001 in both sexes).

**Table 2.** Daily drinkers of sugar-sweetened beverages according to demographic, dietary and lifestyle characteristics, by sex in the Tromsø study: *Fit Futures*.

**Table 2.** *Cont.*

1 All participants are used to calculate the percentage of daily drinkers, but the numbers for the non-daily drinkers have been suppressed for better legibility. 2 The n is the number of daily drinkers/the total number of participants reporting the given characteristic. 3 Chi-square test. 4 Sum of four different milk types (different fat content) and yoghurt. Includes both plain and fermented milk. 5 Weighted mean of weekends and weekdays.

Among females eating less than 0.6 servings of fruits and vegetables daily, there were 40.2% daily drinkers of SSB, compared to 27.1% among those eating 2.3 servings or more (*p* = 0.008). The corresponding numbers for males were 69.5% and 57.0% (*p* = 0.05). Both among males and females, those who drank a glass or more of light/artificially sweetened beverages or fruit juice daily, also had a higher tendency to drink SSB daily. There were differences in prevalence of daily drinkers of SSB of 20–30 percentage points between the highest and lowest categories of these beverages for both sexes. Similar, but smaller, differences were found for more frequent consumption of sweets and snacks. In both sexes, the higher the intake of water, the lower the percentage of daily SSB drinkers (*p* < 0.02). There was no association between drinking SSB and milk consumption.

Rarely eating breakfast or bringing lunch to school was associated with daily SSB drinking, with 20–40% higher prevalence of daily SSB drinking than among those eating these meals every day (Table 2). No association was found between frequency of having dinner and daily SSB drinking. Weight class (thin/normal vs. overweight/obese) was not associated with daily SSB drinking, neither in females nor males. Those not physically active were more often daily drinkers of SSB than the

more active, though the distributions differed between sexes, and the difference was only borderline significant in males. Higher daily screen time was associated with daily SSB drinking in females (*p* = 0.002), but not in males. Smokers and snuff users were more likely to be daily SSB drinkers. Among males, never drinkers of alcohol were less likely to be daily SSB drinkers than more frequent alcohol drinkers, among females, there was no significant association between alcohol drinking and daily SSB drinking in univariate analyses.

In the adjusted model, students in vocational studies had a 59% higher chance of drinking SSB daily compared to those in general studies (only borderline significant for females) (Table 3). Daily drinking of SSB was associated with several dietary variables: eating snacks weekly vs. not (females OR 3.67, 95%CI 2.03–6.64, males OR 2.03, 95% CI 1.29–3.19), drinking light/artificially sweetened beverages (>1 glass daily vs never—females OR 4.05, 95%CI 2.22–7.36, males OR 2.20, 95% CI 1.30–3.72). The same was seen for fruit juice, those drinking a glass or more daily had an odds ratio of daily SSB drinking of 2 or more, compared to never drinkers (significant for both sexes). Not eating breakfast was associated with higher odds of daily drinking of SSB compared to eating everyday (females OR 4.03, 95% CI 1.88–8.63, males OR 2.02, 95% CI 0.97–4.22). Other health behaviours were also associated with daily SSB drinking: A significant association with daily snuffing (females OR 4.93, 95% CI 2.40–10.14, males OR 2.18, 95% CI 1.14–4.16), and the same tendency, although not statistically significant, was seen for daily/occasional smoking. Never drinkers of alcohol had a higher OR of daily SSB drinking than those drinking alcohol twice a month or more often, at least in females (females OR 2.59, 95% CI 1.19–5.66, males OR 1.60, 95% CI 0.84–3.03). Water consumption was no longer a significant covariate. The adjusted model explained 40.8% of the variation in daily drinking of sugar-sweetened beverages for females and 20.6% for males. However, there was no interaction with sex (all *p*-values for interaction >0.08).

**Table 3.** Characteristics Associated with being a daily drinker of sugar-sweetened beverages in the Tromsø study: *Fit Futures*.

**Table 3.** *Cont.*

Mutually adjusted logistic regression analyses. Ref = reference category, OR = odds ratio, CI = confidence interval.

In the model including sex and mother's education, males had 3.74 times higher odds of being daily SSB drinkers compared to females (95% CI 2.68–5.22) (Figure 2). Compared to students with mothers with the longest education, those with mothers with short (OR 1.74, 95% CI 1.11–2.72) or medium length of education (OR 2.06, 95% CI 1.24–3.43), had higher odds of being daily SSB drinkers. Else the results were similar to the main model, with OR estimates between what was found for females and males, but estimates for smoking and alcohol were stabilized. This model explained 37.6% of the variation in daily SSB drinking. There was no interaction with sex, (all *p*-values for interaction >0.12).

**Figure 2.** Odds for being a daily drinker of sugar-sweetened beverages in the Tromsø study: *Fit Futures*. Mutually adjusted model including sex and mother's education, *n* = 870.

#### **4. Discussion**

We found high prevalence of daily SSB drinking in this group of adolescents from Northern Norway. Socio-economic factors (study programme, mother's education), sex, diet (snacks, breakfast), other drinking habits (light/artificially sweetened beverages, fruit juice), and health-related habits (smoking, snuffing, alcohol drinking) all contributed to explaining the variation in daily SSB drinking. The model explained more of the variation in daily SSB drinking in females than in males.

In our study, there were more than twice as many daily drinkers as in the edition of HBSC that took place in the same period and age group, 61% of the males and 32% of the females vs. 25 and 11% [11,15]. We included two questions (carbonated and non-carbonated SSB) rather than one, but it is not likely that this, and differences in response categories, explains the difference in prevalence, but rather that there are differences in intake between the two studies. The HBSC study included a lower number of students from the Northern region, but they used a cluster sampling to ensure a nationally representative sample [11], how this might have influenced the results is uncertain. A previous study from Northern Norway found that among boys, as many as 52.3% of eighth graders and 74.0% of tenth graders were daily drinkers of carbonated SSB, corresponding numbers for girls were 42.1% and 46.8% [23]. In addition, 48.9% of the boys and 45.0% of the girls drank other soft drinks ("squash") daily. That survey took place five years before our study. As we found that 61% of the males and 32% of the females were daily drinkers (of carbonated and non-carbonated SSB combined), this might support the decreasing trend in SSB intake seen over time in the HBSC study [11]. However, the number of daily drinkers still is higher than what is reported in national studies and studies from other parts of Norway. Some of this difference can be explained by differences in age groups and questions included in the different studies, but not all. While our questions covered more than HBSC, we did not include energy/sports drinks, so our prevalence estimates could still be underestimated. Our participants represented urban and rural adolescents in Northern Norway. We excluded some students due to age, and some due to missing variables, but with an exceptionally high participation rate, we still think that the results are representative for adolescents in the region.

Conflicting results exists concerning the stability of SSB consumption. The Oslo Youth survey found moderate to high stability of soft drink consumption from age 15 to 25, but low stability from age 15 to 33 [18]. The FVMM study on the other hand observed a decrease in SSB consumption, and an increase in consumption of artificially sweetened beverages from age 11 to 26 [28]. Hence, continued monitoring of our participants would be interesting in order to gain more knowledge about the stability of the SSB intake.

Higher or more frequent intake in boys than in girls has been reported in several studies [17,29–32]. However, some of the other results were surprising; we did not see associations with BMI, sedentary behaviour, or physical activity in the final models. A lack of association between BMI and consumption of SSB has also been reported in other studies [29]. One study found no association in cross-sectional analyses, but higher BMI in SSB consumers in longitudinal analyses [30]. The same study found associations both cross-sectionally and longitudinally for percentage body fat. A review on confectionery consumption and overweight/obesity found that in cross-sectional studies, obese and overweight children had lower confectionary intakes than normal weight children [33]. In addition, they reported no association in longitudinal studies. Our study is a cross-sectional study, so no causal inferences can be drawn. It is possible that those with high BMI underreport their SSB consumption, or actually reduce their consumption in order to lose weight [29]. Alternatively, BMI might not be the best measure of body fatness, or this could be a true association. Spending much time on sedentary activities could also mean spending much time on homework, and need not be only a negative feature.

One could assume that more frequent consumption of one drink would be mirrored by lower/less frequent consumption of other drinks, but daily drinking of SSB was associated with higher consumption of juice. The picture was more mixed for artificially sweetened soft drinks, however, daily drinkers were more frequently never consumers of alcohol, at least among females. In the univariate analyses, there was less SSB consumption among those that drank water more frequently. This association was not significant in the adjusted analyses. Replacing SSB with water would be the healthiest option, but interventions have only produced medium size increases in water consumption [6]. Longitudinal analyses of stability and change across the spectrum of beverages would provide interesting complimentary data. Those rarely eating breakfast had a very high odds for daily SSB-drinking. For this group, availability of healthy options in schools might be an issue.

A recent publication with data from the Nordic countries in the HBSC study found no socioeconomic differences in SSB drinking, as measured by the family affluence scale [11], however, our study suggested that both own educational choice and mother's education were associated with daily SSB drinking, in line with other studies [12,17,20,28–30,32,34]. To the best of our knowledge, this is the first Norwegian study to look at school programme, an indicator of the students own socioeconomic status, in association with SSB consumption. If longitudinal studies can confirm that school programme is a predictor of SSB consumption, targeted interventions in vocational school programmes might be an important avenue for further reducing the SSB consumption. Not living with parents did not influence SSB drinking, but the group was small, so no strong conclusions can be drawn.

The HELENA study assessed the beverage consumption among European adolescents in eight countries [31]. SSB was the second most frequently consumed beverage, after only water, and 55% of the 15–17.5 year olds reported drinking SSB (including sports drinks) during the two days the study covered, compared with our 61% daily drinkers among males and 32% among females. In half the countries studied, SSB were the largest contributor to energy intake among all beverages.

SSB are major contributors to sugar intake in Norwegian adolescents [35], and dietary patterns characterized by high sugar and fat content have been associated with adiposity later in adolescence [36]. The current national plan of action for a better diet aims at halving the number of 15-year-olds that drink SSB 5 times a week or more often [37]. Continued monitoring, and interventions targeting SSB consumption in general, and in males, students in vocational programmes and Northern Norway in particular are warranted, in order to accomplish that goal. Reviews have suggested that education programmes and changes in school environment (restricted access) will reduce intake, but also that substitution of SSB with low sugar/artificially sweetened beverages will reduce sugar and energy intake [38,39]. This is in line with children's own opinions [40]. Restricting access at home and ensuring good parental modelling is another possible avenue [20,22,38,40]. A recent review showed only small decreases in SSB consumption in adolescents after interventions [6], specific analyses on most successful intervention settings could not be done in adolescents, but for children, home-based interventions and modelling/demonstrating the behaviour seemed to improve intervention effects.

This study has several strengths, a fairly large, population-based sample, a very high participation rate, and assessment of a range of covariates that might confound analyses of SSB intake and health. The sample was homogenous with respect to age, and included both rural and urban participants. Taken together, this suggests that our results are representative for adolescents in Northern Norway. Separate questions were asked for both carbonated and non-carbonated SSB, as this better captures the range of products that can be grouped as SSB [41].

The study also has several limitations. The questions about SSB used in this study have not been used in other studies in adolescents and, in general, most of the studies cited here have used different definitions or response categories, hampering comparisons between studies. For instance, not all studies distinguish between carbonated and non-carbonated SSB, and many do not include sports drinks (and neither did ours). Not including sports drinks/energy drinks in the definition probably means that frequency of intake reported here is underestimated. Lack of comparability is a recognized problem, and a recent review has suggested methods that should be used in future studies [41].

The most frequently reported intake category in our study was wide (1–6 glasses per week), and created possibilities for misclassification when combining the data for carbonated and non-carbonated SSB into one variable. Some studies distinguish weekday and weekend consumption, as this may differ considerably [17], but we did not have that information. Consequently, some of the participants

we labelled "daily SSB drinkers" might have consumed larger amounts of SSB on one or two days, rather than one (or more) glass daily. The total amount of sugar and energy ingested will be the same, independent of how the consumption is distributed, but the length of time the teeth are exposed will affect dental caries risk.

Furthermore, the food/drink questions have not been validated. A review on validity of food frequency questionnaires used in adolescents showed that not assessing portion size, shorter time span, medium length, and administration directly to the adolescent, not via a parent gave the best validity [42]. Our questionnaire did not specify the time-span for the food questions, but the other factors were fulfilled. Our study only included participants from Northern Norway, and the results might not be representative for the rest of the country.

As cutting down on sugar intake has been official policy during the last years, and initiatives have been taken to reduce consumption of SSB [13], social desirability bias, i.e., underreporting consumption of products the health authorities discourage, could be an issue. Given the high consumption reported, this seems less likely, but it cannot be ruled out. Although the sample was relatively large, the skewed distribution of some of the variables led to large uncertainty in some of the estimates. The study was cross-sectional, so it is not possible to make causal inferences. For some reason, the variables explained the variation in SSB intake in females better than in males. We lacked information regarding, e.g., individual choices vs. family or peer influence, and availability, or money to purchase SSB [20,21,34,40]. Perhaps such variables are more important for males' food choices than females'. Females reach puberty earlier and have a stronger body focus, and possibly also health focus than males [43,44], this may explain some of the sex differences we found.

#### **5. Conclusions**

In conclusion, particularly males are frequent drinkers of SSB, despite efforts at reducing consumption in recent years. In both males and females, higher prevalence of daily drinking was found than in a national survey. Our study has identified several factors associated with daily SSB drinking, many of them are other unhealthy lifestyle choices, and are important to adjust for in analyses of SSB intake and health. If these associations are confirmed using stronger study designs, these factors could be targeted together in future comprehensive intervention studies. The higher consumption of SSB in vocational studies is of particular concern, suggesting that socio-economic differences in diet start early.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/11/2/211/s1, Supplementary File 1: Dietary questions used in the Tromsø study: *Fit Futures 1*.

**Author Contributions:** Conceptualization: G.S. and V.S.; data curation: V.S. and G.G.; formal analysis: G.S.; funding acquisition: G.G.; investigation: G.G.; methodology: G.S.; project administration: G.S. and G.G.; resources: G.G.; supervision: G.S.; writing—original draft: G.S., V.S., and G.G.; writing—review and editing: G.S., V.S., and G.G.

**Funding:** The Tromsø study: *Fit Futures 1* was set up with contributions from Helse Nord Infrastrukturmidler, UNN Forskningsposten, Helsefak UiT, Troms fylkeskommune, Odd Berg medisinske forskningsfond, Sparebankens gavefond, Simon Fougner Hartmanns Familiefond, and Eckbos legat. The funding bodies had no role in the design, analysis, or interpretation of this paper. The publication charges for this paper have been funded by a grant from the publication fund of UiT The Arctic University of Norway.

**Acknowledgments:** We wish to thank all the adolescents who participated in the Tromsø study: *Fit Futures 1*. Also, we want to thank everyone who contributed to setting up the Tromsø study: *Fit Futures*, and to the data collection, particularly the Clinical Research Department at the University Hospital of North Norway.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

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