Almond supplementation in the absence of dietary advice significantly reduces C-reactive protein in subjects with type 2 diabetes

journal of functional foods 10 (2014) 252–259

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Almond supplementation in the absence of dietary advice significantly reduces C-reactive protein in subjects with type 2 diabetes Karen L. Sweazea *, Carol S. Johnston, Kristin D. Ricklefs, Katherine N. Petersen School of Nutrition and Health Promotion, Arizona State University, Phoenix, AZ, USA

A R T I C L E

I N F O

A B S T R A C T

Article history:

Heart disease and stroke are primary causes of morbidity and mortality among people with

Received 18 February 2014

type 2 diabetes (T2D). The objective of this 12-week randomized, parallel-arm controlled study

Received in revised form 8 May 2014

was to determine if almond supplementation (1.5 oz/d) without further diet instruction im-

Accepted 18 June 2014

proves diabetic and cardiovascular risk markers in individuals with T2D (hemoglobin A1c

Available online

between 6.5 and 9.0%) who were not taking insulin (n = 10) compared to matched controls who were instructed to maintain their customary diet (n = 11). Subjects in the almond-

Keywords:

treated group tended to consume fewer carbohydrates (p = 0.073). There were no signifi-

Almond

cant differences in biomarkers of glucose regulation or oxidative stress; however, the

Diabetes

inflammatory biomarker C-reactive protein was significantly reduced in the almond-

Inflammation

treated group versus controls (−1.2 vs. +4.33 mg/L, p = 0.029). Daily almond ingestion in the

CRP

absence of other dietary or physical activity modification is beneficial in reducing inflam-

Diet

mation in individuals with T2D. Funding was provided by The Almond Board of California. © 2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Tree nuts are a remarkable food as they are a compact source of numerous healthful nutrients including monounsaturated fat, α-linolenic acid, protein, arginine, fiber, vitamin E, magnesium, and copper, as well as an array of healthful phytochemicals such as phytosterols, flavonoids, and proanthocyanidins (Bolling, Chen, McKay, & Blumberg, 2011; King, Blumberg, Ingwersen, Jenab, & Tucker, 2008; Li, Yao, & Siriamornpun, 2006). The low glycemic index of tree nuts is a desirable quality when determining appropriate snack options for individuals with diabetes (Chen, Lapsley, & Blumberg, 2006). In fact, epidemiological evidence consistently links regular nut consumption with reduced risk for heart disease, diabetes,

cancer, and obesity (Bao et al., 2013; Martínez-González & Bes-Rastrollo, 2011; Pan, Sun, Manson, Willett, & Hu, 2013; Sabaté & Wien, 2010). The evidence for protection against heart disease is the most robust, and the U.S. Food and Drug Administration approved a qualified health claim for nuts and heart disease in 2003: Scientific evidence suggests but does not prove that eating 1.5 ounces per day of most nuts as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease (FDA, 2013). Interventional trials also suggest beneficial effects of nut intake for improving biomarkers associated with risk for cardiovascular disease and diabetes in adults (Jenkins et al., 2008a; Li et al., 2011; Wu et al., 2014). For example, Jenkins et al. (2008a) showed that hyperlipidemic subjects who consumed 70 g of almonds daily for 4 weeks had lower urinary insulin secre-

* Corresponding author. Tel.: +1 480-965-6025; fax: +1 480-968-4399. E-mail address: [email protected] (K.L. Sweazea). Abbreviations: CRP, C-reactive protein; HbA1c, hemoglobin A1c; 4-HNE, 4-hydroxynonenal; HOMA-IR, homeostatic model assessment of insulin resistance; IL-6, interleukin-6; T2D, type 2 diabetes; TNFα, tumor necrosis factor alpha http://dx.doi.org/10.1016/j.jff.2014.06.024 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

journal of functional foods 10 (2014) 252–259

tion, a marker of improved insulin regulation. Similarly, Li et al. (2011) showed that consumption of 60 g of almonds daily in combination with a low fat diet for 12 weeks significantly improved biomarkers of diabetes and lowered inflammation and oxidative stress markers. However, the daily nut portion in some of these trials is greater than twice the amount customarily consumed by individuals in natural settings (as high as 60– 73 g/d, which equates to about 2–2.6 oz/d). Jenab et al. (2006) collected dietary data from over 35,000 individuals from 10 European countries and determined that the typical portion size for nuts was 15–35 g/d. Moreover, many interventions required participants to follow strict dietary protocols and instructed participants to reduce food consumption to accommodate the high energy content of the added nuts. Hence, it is difficult to assess the external validity of the evidence and the value of recommending nut ingestion to the general public. The antioxidant properties of almonds in particular are welldocumented and attributed to their relatively high content of vitamin E and polyphenols (Chen et al., 2006). Many of these polyphenols and antioxidant properties are found in the skin of almonds (Garrido, Monagas, Gómez-Cordovés, & Bartolomé, 2008). In a small pilot trial, individuals with well-controlled type 2 diabetes displayed a modest but significant reduction in hemoglobin A1c (HbA1c) from 7.1 ± 0.2% to 6.8 ± 0.3% (p = 0.045) after 12 weeks of ingesting 1 oz (28 g) whole almonds on five days per week without any other diet instruction or alteration. In addition, body mass index was reduced 4% with almond consumption (p = 0.047) (Cohen & Johnston, 2011). These data suggest that almond ingestion, at a typical portion size and without other dietary modification, may be a feasible and effective diet tactic for individuals with type 2 diabetes (T2D). The portion size used in this pilot study is also more representative of typical portion sizes in contrast to the large portions used in other studies. Therefore, these findings present realistic results that can be expected with typical almond consumption in subjects with well-controlled T2D (Jenkins et al., 2008a; Li et al., 2011; Wu et al., 2014). The purpose of this study was to expand on these results and examine the efficacy of almond ingestion for 12 weeks (at the FDA recommended dose of 1.5 oz almonds daily on 5–7 days per week with no further diet instruction or alteration) for improving glucose regulation, lipid profiles, and blood pressure, and reducing markers of oxidative stress and inflammation in adults with T2D that was not well-controlled.

2.

Methods and materials

2.1.

Participants

Healthy adults, 25–75 years of age and diagnosed by a physician with T2D for at least 6 months, were recruited from the Phoenix metropolitan area in 2012 and 2013. The exclusion criteria were a history of peanut and/or tree nut allergy, insulin use, hemoglobin A1c (HbA1c) <6.5% or >9.0%, dietary intake of >12% monounsaturated fatty acids, active disease states (other than diabetes), anticipated change in diet or physical activity levels, and pregnancy or lactation. Subjects currently taking prescription medications, including oral hypoglycemic agents (other than insulin), statins and hypertensive medications were

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instructed to maintain consistent use throughout the study. Written informed consent was obtained from all participants prior to the start of the trial, and the study was approved by the Institutional Review Board at Arizona State University. Of the 155 volunteers who responded to a pre-screening survey to determine eligibility for participation, 73 were not eligible because they did not have T2D (n = 26), did not respond to emails (n = 11), were prescribed insulin (n = 24), or reported HbA1c values less than 6.3% (n = 12). Of the 82 who were invited for HbA1c screening, 58 were excluded for having HbA1c outside the range of the inclusion criteria (6.5–9.0%); the remaining 24 volunteers were enrolled in the study.

2.2.

Experimental design

Study participants were instructed to reduce all nut consumption at least one week prior to and during the 12-week trial to two or less servings of non-trial nuts weekly. Participants completed a validated physical activity questionnaire (Godin & Shephard, 1985) at baseline as well as a 3-day diet record at baseline and trial weeks 6 and 12. Participants were stratified by HbA1c, age, duration of diabetes, gender, and body mass index and randomly assigned to the almond (n = 12) or control (n = 12) groups. Participants in the almond group were instructed to consume 1.5 oz (43 g) almonds 5–7 times weekly and to maintain their routine diet and activity patterns. Participants in the control group were similarly instructed to maintain their typical diet and activity patterns during the trial. Control participants received a 12-week supply of almonds at the conclusion of the trial. Whole trial almonds were supplied by The Almond Board of California and sealed in 1.5 oz airtight packaging (1.5 oz portion: 260 kcal, 6.9 g carbohydrate, 21.9 g fat), and a 6-week supply of almonds was given to participants in the almond group at baseline and study week 6. Nutrition labels were not provided on the packaging to avoid intentional alterations in the subject’s normal dietary habits. At each visit (baseline and study weeks 6 and 12), blood pressure was measured using a digital monitor (Medline Automatic Digital BP monitor, Medline Industries Inc, Mundelein, IL) and body mass (weight in light clothing) and percent body fat were assessed (Tanita scale, Model TBF-300A, Tanita Corp, Arlington Heights, IL). Waist circumference was measured at the umbilicus using a flexible tension tape at each visit. A fasting venous blood sample (no food or drink with the exception of water for >10 hrs) was also collected at each visit. Participants were told to avoid exercise on the day prior to and the morning of these visits.

2.3. Biomarkers of glucose regulation, blood lipids, oxidative stress and inflammation Plasma samples were collected using vacutainers coated with either heparin (vitamin E analyses only) or EDTA (all other analyses) and centrifuged at 3000 rpm for 15 minutes at 2–4 °C to separate formed elements from plasma. Plasma was aliquoted into separate tubes for each assay to avoid degradation and stored at −80 °C until analyses. Fasting blood samples at study baseline and week 12 were analyzed for total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, C-reactive protein (CRP) and serum glucose

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using the point of care COBAS C111 chemistry random access autoanalyzer (Roche Diagnostics, Indianapolis, IN). Lipids and CRP were measured in duplicate by adding 200 µL plasma to a sample cup (Cat. No. 5085713001, Roche Diagnostics) and programming the analyzer to measure each variable using specific reagents. Lipids were measured by enzymatic colorimetric assays with specific reagents from Roche Diagnostics for total cholesterol (CHOL2, Cat. No. 04718917190), triglycerides (TRIGL, Cat. No. 04657594190), HDL cholesterol (HDLC3, Cat. No. 04657560190), and LDL cholesterol (LDL C, Cat. No. 04657578190) whereas CRP was measured in the samples by particle enhanced turbidimetric assays (CRPLX, Cat. No. 04657497190). Glucose was measured in duplicate by adding 200 µL plasma to separate sample cups and selecting the glucose UV test (GLUC2, Cat. No. 04657527190). The COBAS C111 analyzer was calibrated daily for each assay. According to an analysis by the Laboratory Corporation of America, the COBAS C111 autoanalyzer met or exceeded expectations for precision, linearity, and reliability and the performance was rated as world class by sigma metrics analyses (Bowling & Katayev, 2010). HbA1c was measured on 1 µL aliquots of whole blood using a point of care immunoassay autoanalyzer with self-contained immunoassay cartridges that measure monoclonal antibody agglutination reactions (DCA Vantage, Siemens Healthcare Diagnostics Inc., Deerfield, IL). The DCA Vantage is calibrated daily before use. According to a recent study, the DCA Vantage system passed the National Glycohemoglobin Standardization Program (NGSP) with the clinically relevant range of <3% coefficient of variation (Lenters-Westra & Slingerland, 2010). Serum insulin concentrations were measured by radioimmunoassay using a commercially available RIA kit (Cat. No. HI14 K; Millipore, St. Charles, MO). Samples were assayed according to the manufacturer’s protocol. Serial dilutions of the supplied human insulin standard were prepared (0, 3.125, 6.25, 12.5, 25, 50, and 100 µU/mL). Assay buffer (100 µL) was added to all 12 × 75 mm borosilicate glass tubes followed by 100 µL of each standard or sample in duplicate. Hydrated 125I-Insulin (100 µL) was then added to each tube followed by 100 µL Human Insulin Antibody. Tubes were vortexed, covered, and allowed to incubate overnight at room temperature. The following morning, 1.0 mL cold (4 °C) Precipitating Reagent was added to each tube. The tubes were then vortexed and incubated for 20 minutes at 4 °C. Tubes were then centrifuged for 30 minutes at 3750 rpm, 4 °C. The supernatant from each tube was decanted and the radioactivity measured using a gamma counter (Wizard 1470 gamma counter, Perkins Elmer, Waltham, MA). Homeostatic model assessment of insulin resistance (HOMAIR) was calculated from the fasting plasma glucose and insulin values using the following equation: fasting glucose (mmol/ L) × fasting insulin (mU/L)/22.5 with higher levels indicative of reduced insulin sensitivity. Vitamin E concentrations were determined by high performance liquid chromatography as previously described (Smith et al., 2011). Plasma was assessed for 4-hydroxynonenal (4-HNE), a metabolite of unstable lipid peroxyl radicals (Devasagayam, Boloor, & Ramasarma, 2003) and a biomarker of oxidative stress using a commercially available human ELISA kit (Cat. No. MBS161454; MyBioSource, San Diego, CA). Per the manufacturer’s instructions, the kit was allowed to equilibrate to room temperature prior to beginning the assay. A standard curve (0, 20, 40, 80, 160,

320 ng/L 4-HNE) was created using the supplied 4-HNE standard solution. A 50 µL aliquot of each biotinylated standard was added in duplicate along with 50 µL of streptomycin-HRP to the respective standard wells in the supplied microplate precoated with anti-human 4-HNE monoclonal antibody. Samples were also analyzed in duplicate by adding 40 µL of sample, 10 µL 4-HNE antibodies, and 50 µL streptavidin-HRP to the respective sample wells. The plate was then sealed and allowed to incubate at 37 °C for one hour. The plate was then washed according to the manufacturer’s instructions and 50 µL each of chromogen solution A and B was then added to each well and the color was developed at 37 °C for 10 minutes in the dark. Stop Solution (50 µL) was then added to each well and the absorbance was read at 450 nm within 10 minutes (Multiskan GO microplate spectrophotometer, Thermo Fisher Scientific, Waltham, MA). Plasma concentrations of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) were measured as additional biomarkers of inflammation. TNFα was measured by ELISA (Cat. No. EH3TNFA; Pierce, Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer’s instructions. Briefly, 50 µL of Sample Diluent was added to each well of a microwell plate pre-coated with anti-human TNFα followed by the addition of 50 µL of each standard (0, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 pg/mL TNFα) or sample in duplicate to the appropriate wells. The plate was then covered and allowed to incubate for one hour at room temperature on a plate rocker. Following three washes with diluted Wash Buffer (prepared according to the instructions provided), 100 µL of Biotinylated Antibody Reagent was added to each well and the plate was again covered and allowed to incubate for one hour at room temperature on a plate rocker. Following a second series of three washes with diluted Wash Buffer, 100 µL Streptavidin-HRP Reagent was added to each well and the plate was covered and allowed to incubate for an additional 30 minutes at room temperature on a plate rocker. Following a third series of washes with diluted Wash Buffer, 100 µL TMB Substrate Solution was added to each well and allowed to develop at room temperature for 30 minutes in the dark (unsealed) at which point 100 µL Stop Solution was added to all wells and the absorbance was read at 450 nm and 550 nm. The absorbance values from 550 nm were subtracted from the values at 450 nm to correct for imperfections on the microwell plate. Interleukin-6 (IL-6) was similarly measured using a commercially available ELISA (Cat. No. HS600B; R&D Systems, Minneapolis MN). Standards were prepared according to the directions in the kit (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0 and 10.0 pg/mL IL-6). Assay Diluent RD1-75 (100 µL) was added to each well of a microplate pre-coated with an anti-human IL-6 monoclonal antibody. Standards and samples (100 µL each) were added in duplicate to the microplate and the plate was covered and incubated for two hours at room temperature on a plate rocker. The plate was then washed with diluted Wash Buffer (prepared according to the manufacturer’s directions) five times after which 200 µL Human IL-6 HS Conjugate was added to each well. The plate was again covered and allowed to incubate for two hours at room temperature on a plate rocker. Following a second wash as described above, 50 µL Substrate Solution was added to each well and the plate was again covered and allowed to incubate for 60 minutes at room temperature on the

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Table 1 – Dietary data in control and almond groups at baseline and week 12.

Total energy intake 24 h (kcal) Control Almond Carbohydrate (%) Control Almond Fat (%) Control Almond Monounsaturated fatty acids (MUFA, g) Control Almond Alpha-tocopherol (mg) Control Almond

Baseline

Week 12

Change

p-value

n = 10 n=6

1670 ± 463 2172 ± 438

1590 ± 412 1810 ± 314

−80 ± 553 −362 ± 630

0.313

n = 10 n=6

41 ± 9 47 ± 13

46 ± 9 39 ± 12

5 ± 10 −8 ± 17

0.073

n = 10 n=6

38 ± 11 44 ± 8

37 ± 6 42 ± 11

−0.01 ± 0.09 −0.02 ± 0.13

0.635

n = 10 n=6

14 ± 5 17 ± 6

13 ± 5 24 ± 8

−1 ± 9 7±6

0.147

n = 11 n = 10

2±1 3±1

3±1 10 ± 6

1±2 7±5

0.007

Data are expressed as mean ± SD. Diet records were not complete for five participants (1 control and 4 almond participants). Variable change computed as week 12 value minus baseline value; p-value represents Mann–Whitney U test for change data.

benchtop. Amplifier Solution (50 µL) was then added to each well, the plate re-sealed and allowed to incubate for an additional 30 minutes at room temperature on the benchtop. The color development reaction was stopped with the addition of 50 µL Stop Solution to each well and the absorbance was read at 490 nm.

2.4.

Statistical analyses

Coefficients of variation for sample replicates in all assays were ≤10%. Data were analyzed using PASW Statistics 19.0 (Predictive Analytics SoftWare Statistics package, IBM, 2011). Descriptive data at baseline were assessed using independent t-tests. Change from baseline was reported for outcome variables and differences between groups were identified using the Mann– Whitney U test. Spearman’s Rank correlation was used to identify relationships between variables. All results were expressed as means ± SD, and a p-value ≤0.05 was considered statistically significant.

3.

Results

Three female participants (1 in the control group and 2 in the almond group) withdrew early in the trial citing scheduling conflicts; hence, data are presented for 21 participants, 11 in the control group (4/7 M/F) and 10 in the almond group (5/5 M/F). Gender, age (54.7 ± 8.9 and 57.8 ± 5.6 y for the control and almond groups respectively), and physical activity levels (calculated as metabolic equivalents: 145 ± 286 and 165 ± 302 kcal· kg−1· wk−1 respectively) at baseline did not differ between groups. Prior to the start of the intervention, 3-day diet record analyses indicated that energy (1878 ± 734 and 2171 ± 471 kcal) and fiber (18 ± 15 and 16 ± 8 g) intakes, as well as the macronutrient energy distributions, did not differ between the control and almond groups. It should be noted that diet records were incomplete with 10 submitted for the control group and 6 for the almond group.

Although energy intakes did not differ significantly between groups over the course of the study, both groups reported a reduction in energy consumption at the end of the trial (−80 ± 553 and −362 ± 630 kcal/d for the control and almond groups respectively, p = 0.313) (Table 1). Vitamin E intakes significantly increased during the study in the almond group in comparison to the control group (+7 ± 5 mg and +1 ± 2 mg respectively, p = 0.007) (Table 1). Carbohydrate intake (as percent energy) tended to fall during the study in the almond group in comparison to the control group (−8 ± 17% and +5 ± 10% respectively, p = 0.073). Although dietary records were incomplete for subjects in the almond group, these findings suggest that dietary carbohydrates might have been replaced by almonds for some subjects in the current study. The 12-week change for anthropometric, blood pressure, or plasma lipid measures did not differ between groups (Table 2). Similarly, the 12-week change in biomarkers of glucose regulation (fasting plasma glucose and insulin, HbA1c, and HOMAIR) did not differ between groups (Table 3). CRP was reduced after the 12-week study in the almond group but not in the controls (−1.20 ± 1.70 and +4.33 ± 10.24 mg/L respectively, p = 0.029) (Table 4). These data remained near significant after removal of an outlying value from the control group (−1.20 ± 1.70 and +1.27 ± 3.54 mg/L, p = 0.052). Almond ingestion for 12 weeks did not lower the biomarker of oxidative stress (4-HNE) or the other markers of inflammation (TNFα and IL-6) in comparison to the controls (Table 4). However, 4-HNE was reduced overall at the end of the 12-week study (−3.2 ± 6.7 for all participants combined, p = 0.065; or −3.6 ± 7.7 and −2.7 ± 5.9 ng/L in the control and almond groups respectively). Moreover, the change in IL-6 was markedly related to the change in CRP over the course of the 12 week study (r = 0.865, p < 0.001) as expected since IL-6 is known to regulate CRP expression (Tousoulis et al., 2013).

4.

Discussion

Almond ingestion (1.5 oz on five to seven days per week for 12 weeks) significantly reduced CRP concentrations by nearly

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Table 2 – Anthropometric, blood pressure and lipid profiles of control and almond groups at baseline and week 12.

Body mass (lbs) Control Almond BMI (kg/m2) Control Almond Waist circumference (in) Control Almond Body Fat (%) Control Almond Systolic blood pressure (mmHg) Control Almond Diastolic blood pressure (mmHg) Control Almond Plasma total cholesterol (mM/L) Control Almond Plasma LDL cholesterol (mM/L) Control Almond Plasma HDL cholesterol (mM/L) Control Almond Plasma triglycerides (mM/L) Control Almond Plasma alpha tocopherol (uM/L) Control Almond Plasma doco tocopherol (uM/L) Control Almond Plasma gamma tocopherol (uM/L) Control Almond

Baseline

Week 12

Change

p-value

n = 11 n = 10

202.6 ± 60.3 234.8 ± 45.3

199.7 ± 56.4 234.5 ± 47.0

−2.9 ± 7.6 −0.3 ± 5.3

0.973

n = 11 n = 10

33.5 ± 8.8 37.2 ± 7.8

33.1 ± 8.3 37.2 ± 8.4

−0.4 ± 1.2 0.0 ± 0.9

0.973

n = 11 n = 10

42.5 ± 7.2 46.8 ± 7.3

42.0 ± 6.5 46.8 ± 7.0

−0.5 ± 1.8 0.01 ± 2.4

0.809

n = 11 n = 10

39.5 ± 9.3 41.6 ± 9.8

38.4 ± 8.0 43.2 ± 7.7

−1.1 ± 3.4 1.7 ± 5.1

0.468

n = 11 n = 10

137.9 ± 18.8 143.3 ± 14.9

133.9 ± 16.1 139.8 ± 22.4

−4.2 ± 17.1 −3.5 ± 17.2

0.853

n = 10 n = 10

80.6 ± 15.6 83.9 ± 11.6

80.2 ± 9.6 81.1 ± 11.7

−1.8 ± 9.0 −2.8 ± 10.9

0.739

n = 10 n = 10

4.21 ± 0.85 4.24 ± 0.87

4.11 ± 0.88 4.34 ± 1.20

−0.10 ± 0.58 0.09 ± 0.56

0.739

n = 10 n = 10

2.58 ± 0.78 2.64 ± 0.83

2.52 ± 0.76 2.71 ± 0.83

−0.05 ± 0.53 0.07 ± 0.55

0.853

n = 10 n = 10

1.03 ± 0.25 1.20 ± 0.15

1.01 ± 0.23 1.25 ± 0.19

−0.02 ± 0.11 0.05 ± 0.08

0.105

n = 10 n = 10

1.93 ± 1.35 1.48 ± 0.41

2.08 ± 1.27 1.53 ± 0.67

0.15 ± 0.37 0.05 ± 0.34

0.529

n = 11 n = 10

2.95 ± 1.04 3.25 ± 0.95

3.15 ± 1.11 3.36 ± 1.00

0.09 ± 0.42 0.05 ± 0.32

0.809

n = 11 n = 10

0.14 ± 0.16 0.23 ± 0.32

0.32 ± 0.44 0.07 ± 0.07

0.19 ± 0.46 −0.16 ± 0.30

0.114

n = 11 n = 10

0.25 ± 0.30 0.12 ± 0.09

0.23 ± 0.16 0.14 ± 0.14

−0.02 ± 0.23 0.02 ± 0.12

0.756

Data are expressed as mean ± SD. Variable change computed as week 12 value minus baseline value; p-value represents Mann–Whitney U test for change data.

Table 3 – Biomarkers for glucose regulation in the control and almond groups at baseline and week 12.

Fasting plasma insulin (pM/L) Control Almond Fasting plasma glucose (mM/L) Control Almond HbA1c (%) Control Almond Homeostatic model assessment of insulin resistance (HOMA-IR) Control Almond

Baseline

Week 12

Change

p-value

n = 10 n = 10

209 ± 191 176 ± 87

226 ± 182 183 ± 94

16.2 ± 168 7.20 ± 54.6

0.481

n = 10 n = 10

8.52 ± 2.21 8.52 ± 2.30

8.58 ± 4.20 7.84 ± 1.61

0.06 ± 3.57 −0.68 ± 1.67

0.796

n = 10 n = 10

7.22 ± 0.91 7.02 ± 0.70

7.05 ± 0.90 6.85 ± 0.75

−0.19 ± 0.73 −0.17 ± 0.32

0.684

n = 10 n = 10

10.8 ± 5.6 11.4 ± 7.0

16.4 ± 18.4 11.2 ± 7.3

5.6 ± 18.2 −0.2 ± 3.3

0.684

Data are expressed as mean ± SD. Variable change computed as week 12 value minus baseline value; p-value represents Mann–Whitney U test for change data.

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journal of functional foods 10 (2014) 252–259

Table 4 – Biomarkers of oxidative stress and inflammation in the control and almond groups at baseline and week 12. Baseline 4-HNE (ng/L) Control Almond IL-6 (pg/mL) Control Almond TNFα (pg/mL) Control Almond CRP (mg/L) Control Almond

Week 12

Change

p-value

n = 11 n = 10

82.6 ± 14.5 84.1 ± 15.7

79.0 ± 10.0 81.4 ± 13.2

−3.6 ± 7.7 −2.7 ± 5.9

0.863

n = 11 n = 10

1.61 ± 1.27 1.28 ± 0.59

1.81 ± 1.97 1.01 ± 0.58

0.20 ± 1.37 −0.12 ± 0.27

0.314

n = 11 n = 10

27.5 ± 8.1 33.8 ± 10.4

28.5 ± 7.6 35.0 ± 9.8

1.0 ± 4.4 1.3 ± 4.8

0.756

n = 10 n = 10

5.87 ± 6.67 4.31 ± 3.75

10.20 ± 16.6 3.11 ± 2.49

4.33 ± 10.24 −1.20 ± 1.70

0.029

Data are expressed as mean ± SD. Variable change computed as week 12 value minus baseline value; p-value represents Mann–Whitney U test for change data.

30% in individuals with type 2 diabetes whereas CRP concentrations were not reduced in matched controls with no dietary change (p = 0.029 for interaction). However, almond ingestion did not influence biomarkers of glucose control, blood lipids, or oxidative stress, nor did almond consumption modify adiposity or blood pressure after 12 weeks in comparison to the control treatment. As expected, subjects in the current study had higher than normal baseline plasma glucose concentrations (healthy: <5.49 mM/L; American Diabetes Association, 2012), HbA1c concentrations (healthy: 4.0–5.6%, Kratz, Pesce, Basner, & Einstein, 2012), and insulin concentrations (30–90 pM/L; Millipore), which are characteristics of diabetes. Baseline concentrations for TNFα for subjects in the current study were higher than mean values reported for healthy individuals by the manufacturer of the ELISA (2.45 pg/mL; Thermo Scientific) as expected with obesity and diabetes. Plasma lipids at baseline were slightly higher than optimal ranges for LDL cholesterol and triglycerides (LDL <2.59 mM/L, HDL 1.04–1.55 mM/L, total cholesterol <5.18 mM/ L, triglycerides <1.70 mM/L; Kratz et al., 2012), characteristics of obesity. Subjects in the current study also showed signs of vitamin E deficiency as plasma concentrations at baseline were lower than reported for healthy individuals (11.6–41.8 µM/L; Kratz et al., 2012). IL-6 concentrations measured for subjects in the current study were similar to mean values reported for healthy individuals by the kit manufacturer (1.49 pg/mL; R&D Systems) and CRP concentrations were within the normal range for assessing inflammation in healthy subjects (<10.0 mg/L; Kratz et al., 2012). Reference ranges for 4-HNE are not currently available. To date, there have been three published trials examining the efficacy of almond ingestion in individuals with T2D: two trials followed strict diet plans incorporating large amounts of almonds (>60 g/d) and one trial asked participants to consume a serving of almonds daily (28 g) without further diet modification or instruction. For the two trials with strict diet plans, the results were equivocal. Lovejoy, Most, Lefevre, Greenway, and Rood (2002) incorporated 57–113 g almonds into the daily diets of 30 individuals with T2D for four weeks using a randomized crossover design. All foods were provided to participants, and two diet plans (energy adjusted for weight maintenance) were tested (high-fat [37% energy] and low-fat

[25% energy]). Although plasma lipids were favorably impacted, almond ingestion did not influence any of the glucose or insulin measures. Li et al. (2011) conducted a 12-week randomized crossover trial in 20 Chinese adults with T2D who were placed on low-fat diet plans (energy adjusted for weight maintenance) with 20% energy from almonds (~60 g/d). The almond diet was associated with modest reductions in fasting insulin (−4%) and glucose (−0.8%) concentrations and HOMA-IR values (−9.2%). Plasma lipids and markers of inflammation and oxidative stress were also favorably influenced by almond ingestion. In a recent study, Choudhury, Clark, and Griffiths (2014) found that 50 g of almonds per day for 4 weeks significantly increased plasma vitamin E concentrations and improved blood pressure but did not affect plasma lipids or biomarkers of oxidative stress in healthy men. Hence, these trials indicate that high intakes of almonds favorably influence markers of heart disease, corroborating the epidemiological data; however, the benefits of almond ingestion for improving the diabetic condition are not clear. Furthermore, these trials used high doses of almonds and strict dietary control was necessary to ensure weight maintenance. Such a protocol may be impractical and unsustainable for free-living adults. Cohen and Johnston (2011) examined the effect of one serving of almonds (28 g/d; 173 kcal) versus cheese sticks (160 kcal) on fasting glucose and insulin concentrations and HbA1c in individuals with T2D. No other diet modification was made. After 12 weeks, almond ingestion, as compared to cheese, significantly reduced HbA1c (−4%) and body weight; yet fasting glucose and insulin concentrations did not change. These data suggest a possible benefit for recommending almonds as snacks for individuals with diabetes. A similar experimental protocol was followed in the present report, modest daily almond ingestion without further dietary modification, and, although not significant, fasting glucose concentrations fell 8% and HbA1c and HOMA-IR scores fell ~2% in the almond group. Moreover, a significant reduction in CRP concentrations was noted in the almond versus control groups. Elevated CRP is linked with an increased risk of cardiovascular disease in people with T2D (Asegaonkar et al., 2011; Soinio, Marniemi, Laakso, Lehto, & Rönnemaa, 2006). Statin therapy is currently the standard treatment to prevent cardiovascular events in people with diabetes (Eldor & Raz, 2009). However, in a recent study of overweight

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and obese individuals with T2D (Belalcazar et al., 2013), standard statin therapy was only effective at reducing plasma CRP concentrations 14–20% in comparison to the nearly 30% reduction observed in subjects treated with almonds in the current study. Furthermore, the effect size for the almondinduced change in CRP was large (18.4% of variance explained). Therefore, almonds may be an effective, simple strategy to reduce inflammation and cardiovascular disease risks in subjects with poorly controlled T2D. This study was limited by the small sample size (10–11/ group) and incomplete dietary records from subjects in the almond group (n = 6). Based on the outcome data reported herein, the trial had 80% power to detect a 0.4% difference in HbA1c and 80% power to detect a 2.1 mg/L change in CRP. Treatment compliance was supported by a significant increase in dietary vitamin E (+130%, p = 0.007), a modest reduction in dietary energy from carbohydrates (−17%, p = 0.079), and a modest rise in plasma HDL cholesterol (+5%, p = 0.105) in the almond group in comparison to controls. Others report similar changes in diet and plasma profiles for almond intervention trials using much larger dosages of almonds, 60–70 g daily (Jenkins et al., 2008b; Wien et al., 2010). The reduced energy intake in the group that had added almonds to their routine diets (−17%) suggests that almonds may have increased satiety resulting in reduced overall energy consumption. Energy intake decreased less markedly (−4%) in the control group. The 4% reduction in 4-HNE among all participants (p = 0.065) suggests the possibility that both groups may have modified their lifestyle and/or eating habits leading to a reduction in this marker of lipid peroxidation.

5.

Conclusion

The data described in this study suggest that almonds may be a good snack option for individuals with type 2 diabetes since they do not negatively affect blood glucose, oxidative stress or inflammation. Rather, almonds are beneficial at lowering CRP by almost 30% in people with poorly-controlled type 2 diabetes.

Acknowledgements The authors would like to thank Ginger Hook and Matthew Calhoun for technical assistance, Dr. Samer Alanbagy for assistance with recruiting, as well as Dr. Kevin McGraw for vitamin E analyses.

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