Participants and recruitment

The study was conducted between July 2012 and January 2016 at the Cholesterol Research Center (Berkeley, CA). Twenty four men and women were recruited from participants in our previous dietary studies and from the local community through internet and newspaper advertisements, outreach programs, bulk mailing and referrals through collaborations with other academic or corporate institutions. Participants who passed a self-administered web-based pre-screening questionnaire were contacted by a recruiter to review the eligibility requirements and study protocol, and by the study nutritionist to discuss the dietary requirements. Those who agreed to the study protocol received a clinical assessment and blood draw during their initial screening visit. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and the procedures involving human subjects were approved by the Children’s Hospital and Research Center Oakland Institutional Review Board. Written informed consent was obtained from all subjects. This clinical trial was registered on ClinicalTrials.gov under the identifier NCT01792648 (https://clinicaltrials.gov/ct2/show/NCT01792648).

Our initial intent was to study individuals with atherogenic dyslipidemia of the metabolic syndrome, defined by triglycerides ≥1.69 mmol/L, HDL-cholesterol < 1.03 mmol/L (men) or < 1.29 mmol/L (women), and with at least one additional characteristic of metabolic syndrome (waist circumference > 102 cm (men) or > 88 cm (women), blood pressure ≥ 130/≥85 mmHg, or fasting glucose ≥6.1 mmol/L) [14]. Of the screened participants who had triglycerides > 1.69 mmol/L, only ~ 65% had low HDL-cholesterol levels consistent with atherogenic dyslipidemia. Of these, less than 10% had one additional criterion for metabolic syndrome and were willing to take part in the study. Such low prevalence of atherogenic dyslipidemia with features of the metabolic syndrome may be a consequence of our stringent enrollment criteria which excluded individuals taking lipid- or glucose-lowering medications, smokers, and the presence of comorbidities. As such, and after sustained difficulty identifying participants who met these criteria, only abdominal obesity (waist circumference > 88 cm for women and > 102 cm for men) was retained as the selection criterion. Of the 24 participants who completed the study, 2 did not meet the waist criterion but had other characteristics of metabolic syndrome. Exclusion of these two participants from data analysis did not affect any of the parameters measured (lipids, lipoproteins, blood pressure, insulin, glucose, inflammatory markers). Changes to the enrollment criteria were approved by our Institutional Review Board.

Additional screening inclusion criteria included: 1) ≥ 20 years; 2) no history of coronary heart disease, cerebrovascular disease, peripheral vascular disease, bleeding disorder, liver or renal disease, diabetes, lung disease, HIV, or cancer (other than skin cancer) in the last 5 years; 3) not pregnant or breast feeding, and agreeing to use appropriate barrier contraception throughout the study for women of childbearing potential; 4) no current use of hormones or drugs known to affect lipid metabolism or blood pressure; 5) no current use of nicotine products or recreational drugs; 6) willingness to abstain from alcohol or dietary supplements during the study; 7) systolic blood pressure < 160 mmHg and diastolic blood pressure < 95 mmHg; 8) body mass index (BMI) ≤ 38 kg/m2; 9) total- and LDL-cholesterol < 95th percentile for sex and age, 10) fasting triglycerides > 0.56 mmol/L and < 5.65 mmol/L, 11) fasting blood sugar < 7.0 mmol/L ; 12) thyroid stimulating hormone (TSH) within normal range; and 13) weight stable for > 3 months.

Experimental design and setting of the study

The diet intervention was carried out on an outpatient basis with careful monitoring of compliance. In a randomized crossover design, each participant consumed the three experimental diets (a higher-carbohydrate (CHO) reference diet (CHO high ); a higher-CHO diet with almonds (CHO high + almonds ); and a lower-CHO reference diet (CHO low )) for 3 weeks each, separated by 2-week washout periods during which participants consumed their habitual diet (see Additional file 1: Table S1). Diet assignments were kept in sequentially numbered sealed envelopes and assigned to the participant by the clinic staff 1–2 days before starting the intervention. Investigators and laboratory staff were blinded to diet assignment, while clinic staff were not. Participants were not informed of their diet assignment, but due to the nature of the experimental diets, they were likely able to identify them. The three experimental diets consisted of a higher carbohydrate reference diet (CHO high diet: 50% E carbohydrate; 15% protein; 35% total fat); an almond-supplemented (20% E) diet with similar macronutrient composition to the higher carbohydrate reference diet (CHO high + almond diet: 50% E carbohydrate; 15% protein; 35% total fat); and a lower carbohydrate reference diet (CHO low : 25% E carbohydrate; 28% protein; 47% total fat). The CHO high diet followed current dietary recommendations that focus on restricting saturated fat and increasing intake of unsaturated fat (Table 1). The CHO high + almond diet provided 20% E from almonds in partial replacement for cooking oils and other fat sources (avocados, olives, etc.), with all other foods being otherwise comparable among the standard reference and almond-supplemented diets. Almonds (nonpareil variety) were provided by the Almond Board of California. They were distributed to participants in pre-weighed packets when consumed as raw whole unsalted almonds (10% E), with the remaining almonds (10% E) consumed as raw almond meal integrated in customized unit foods (a choice of either roasted pepper almond spread, almond soup or almond cake). For both the CHO high and CHO high + almond diets, the ratio of starch to total sugars was 60/40, with fruit and dairy products constituting the bulk of total sugars, and added sugars representing 6% of daily calories. The composition of the CHO low diet was based on our earlier findings that more extreme carbohydrate restriction, from 54% E to 26% E, is required to significantly reduce LDL-cholesterol as well as small and very small LDL particles, whereas more moderate carbohydrate restriction (39% E) failed to significantly reduce LDL-cholesterol or its component subclasses [15]. For the CHO low diet, the ratio of starch to total sugars was 70/30, with fruit constituting the bulk of total sugars and added sugars representing < 3% of daily calories. During the washout period the participants consumed their usual home diets. Comparable amounts of total fat in the standard reference and almond-supplemented diets were achieved primarily by limiting intake of foods rich in unsaturated fat and partially replacing cooking oils with almonds.

Table 1 Composition of experimental dietsa Full size table

Participants were provided ~ 65% daily energy in the form of two frozen entrees (lunch and dinner) and snacks. In addition, participants received detailed dietary instructions, standardized menus, checklists, and shopping lists for home preparation of breakfast and sides for the remaining food items on the menu. Itemized grocery receipts were collected regularly from participants to verify their purchase of perishable foods on their shopping list. Participants were instructed to eat all food items provided/prescribed, and to report any deviations from the protocol. A compliance score (1–5-point scale, where 5 is indicative of high compliance) was assigned to each study participant by the staff nutritionist, based on menu checklists, itemized grocery receipts and information gathered from weekly interactions.

The nutrient composition of the diets was assessed using ProNutra software (Viocare Technologies, Inc.) and the Nutrition Data System for Research (University of Minnesota). Three-day rotating menus were provided at four calorie levels (2000, 2500, 3000, 3500 kcal) for each of the diets, and snacks (200–350 kcal) similar in nutrient composition to the experimental diets were provided for individuals whose calorie needs were intermediate to the 4 calorie levels available. The participants’ energy requirements for maintaining stable weight were estimated using the Institute of Medicine equation [16]. During the study, participants were required to maintain their body weight within ±3% of their initial weight over the course of any consecutive two weeks.

Height and weight were measured during the clinic visits. Participants wore a pedometer to monitor daily steps. Baseline steps per day were measured during the study run-in period and participants were asked to maintain this level of activity throughout the study. Number of steps and other physical activities were recorded in logs and reviewed during weekly diet visits. Fasting blood samples were collected at the end of each dietary period for measurement of plasma lipids, lipoproteins, lipoprotein subfractions, and apolipoproteins B and AI, as well as markers of insulin resistance and inflammation after fasting overnight for 12–14 h. Plasma was separated immediately by centrifugation at 4 °C. Including the screening visit, participants visited the clinic for a total of 7 blood draws and met with the research nutritionist weekly on 14 separate occasions.

Laboratory measurements

Plasma triglycerides, total- and HDL-cholesterol were measured by enzymatic endpoint analysis on a clinical chemistry analyzer (Liasys 330) using methodology previously described [17,18,19]. Triglyceride and cholesterol measurements are standardized through the CDC-NHLBI lipid standardization program. LDL-cholesterol was calculated from the Friedewald equation [20]. ApoB and apoAI were determined by immunoturbidimetric assay using the ITA reagent kit [21, 22]. Plasma particle concentrations of VLDL, intermediate density lipoprotein (IDL), LDL and HDL subfractions were analyzed by gas-phase electrophoresis (i.e., ion mobility [23, 24]). Inter-assay variations of the subfraction measurements were minimized by the inclusion of two in-house controls in each preparatory process and by duplicate analysis (CV < 15%).

Fasting glucose was measured by enzymatic endpoint analysis and fasting insulin by ELISA (EZHI-14 K Human Insulin ELISA kit; Millipore, Billeria, MA, USA), with two in-house quality control standards. HOMA-IR was calculated as a marker of insulin resistance (insulin (μU/mL) x glucose (mg/dL)/405) [25]. Plasma samples were assayed for a multi-analyte panel of inflammatory biomarkers (interleukins 1, 6, 8, and 10, resistin, TNF-alpha, PAI-1, leptin, MCP, SAA, lipocalin, and BAFF) using a multiplex immunoassay method through a CLIA certified commercial laboratory (Rules Based Medicine Austin, TX).

Statistical analyses