Frequent consumption of nuts, an important component of plant-based diets, is associated with 15% lower total cardiovascular disease (CVD) and 23% lower CVD mortality rates.¹ Small, short-term randomized controlled trials indicate that diets supplemented with nuts have a consistent cholesterol-lowering effect; however, no trials of nut-enriched diets for lipid changes focused on elderly individuals, recruited participants from diverse geographical locations, or lasted 2 years.² Also, there is little information concerning the effects of nuts on lipoprotein subclasses.

We hypothesized that incorporating walnuts into the usual diet would improve the lipid profile irrespective of differences in geographical and dietary background. The WAHA study (Walnuts and Healthy Aging) is a 2-center (Barcelona, Spain and California, USA), 2-year, parallel-group randomized controlled trial testing the effects of walnut-supplemented diets in healthy elderly individuals (URL: https://www.clinicaltrials.gov; Unique identifier: NCT01634841).³ Lipoprotein changes were a prespecified secondary outcome. The study was approved by the Ethics Committee of each center. Data, analytic methods, and study materials will not be made available to other researchers because of Ethics Committees’ restrictions. Eligible candidates were cognitively healthy elders (63–79 years of age) without major comorbidities.³ After providing informed consent, participants (n=708) were allocated to either a walnut-free (control) or walnut-supplemented diet (≈15% of energy, 30–60 g/d). In 2-monthly visits, compliance, tolerance, medication changes, and body weight were recorded. At each visit, 8-week allotments of raw, pieced walnuts were delivered to the corresponding group. Throughout the study, participants were supervised by their primary physicians, who changed medications, including lipid-lowering drugs, according to their assessment of risk factor levels.

Baseline and 2-year fasting plasma glucose, cholesterol, triglycerides, and high-density lipoprotein cholesterol were determined by standard enzymatic methods; low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald formula. Advanced lipoprotein testing was performed with Liposcale, a validated 2-dimensional ¹H-nuclear magnetic resonance spectroscopy, at Biosfer-Teslab (Reus, Spain).⁴ We analyzed 2-year differences in nutrient intake and body weight by 1-way ANOVA and changes in glucose and lipoproteins by multivariable-adjusted ANCOVA.

Results disclosed that 636 participants completed the study (90% retention rate) and 628 had full data for lipoprotein analyses (mean age 69 years, 67% women, 32% treated with statins). Their clinical characteristics did not differ from those of completers for the primary cognitive outcome.³ Mean baseline LDL-C and triglycerides were 117 and 105 mg/dL, respectively. In-trial statin changes were not different by treatment arm. Compliance with the walnut diet was good and body weight was stable, with mean 2-year changes of 0.06 kg (95% CI, –0.32 to 0.44) in the walnut diet and –0.51 kg (95% CI, –0.91 to –0.12) in controls. Reflecting the nutrient composition of walnuts, participants in the walnut group increased intake of energy, total fat, fiber, linoleic acid, and α-linolenic acid (Figure, A). No significant between-group changes in fasting glucose were observed (Figure, B). The walnut diet significantly decreased (mg/dL) total cholesterol (mean –8.5 [95% CI, –11.2 to –5.4]), LDL-C (mean –4.3 [–6.6 to –1.6]), and intermediate-density lipoprotein cholesterol (–1.3 [–1.5 to –1.0]), corresponding to reductions of 4.4%, 3.6%, and 16.8%, respectively, whereas triglycerides and high-density lipoprotein cholesterol were unaffected (Figure, B and C). Total LDL particles and small LDL particle number decreased by 4.3% and 6.1%, respectively (Figure, D). Results were not different by study site. Lipid responses to the walnut diet differed by sex: LDL-C was reduced by 7.9% in men and by 2.6% in women (P-interaction=0.007).

Figure. Mean percent changes at 2 years in dietary variables and lipid and lipoprotein subclasses by intervention group. A, Energy and nutrient intake. B, Measured lipid and lipoprotein cholesterol concentrations. C, Estimated lipoprotein lipid concentrations by nuclear magnetic resonance spectroscopy. D, Estimated lipoprotein particle number by nuclear magnetic resonance spectroscopy. Error bars represent 95% CIs. ALA indicates α-linolenic acid; C, cholesterol; DHA, docosahexaenoic acid; En, energy; EPA, eicosapentaenoic acid; HDL, high-density lipoprotein; LA, linoleic acid; LDL, low-density lipoprotein; MUFA, monounsaturated fatty acids; P, particle number; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TG, triglyceride; and VLDL, very-low-density lipoprotein. *P<0.05. P values for differences in nutrient intake were obtained by 1-way ANOVA. P values for differences in lipids and lipoproteins obtained by ANCOVA adjusted by center, age, sex, body mass index, smoking status (ever smoker/never smoker), APOE ε4 carriership (yes/no), physical activity changes, diabetes (yes/no), dyslipidemia (yes/no), hypertension (yes/no), statin treatment (yes/no), changes in statin doses standardized to simvastatin, and the baseline value of each variable. The estimated marginal means of the changes were used to calculate percent changes from baseline in both groups.

The results of this 2-year randomized controlled trial demonstrate that incorporating daily doses of walnuts (≈15% of energy) to the habitual diet of free-living elderly individuals with an essentially normal lipid profile resulted in a mean 4.3 mg/dL LDL-C reduction, which is modest, although greater responses have been observed among individuals with hypercholesterolemia.² Our data also support a beneficial effect of the walnut diet on nuclear magnetic resonance–assessed lipoprotein subfractions, with reductions of intermediate-density lipoprotein cholesterol (a sizable contributor to remnant cholesterol) and total LDL particles. Prospective studies have reported that LDL particle number consistently outperforms LDL-C in CVD risk prediction and that remnant cholesterol causally relates to CVD independent of LDL-C.⁵ That lipid responses were not different in the 2 cohorts consuming diverse diets strengthens the generalization of our results. WAHA is the largest and longest nut trial to date, overcoming the limitations of prior smaller and shorter nut studies. The novel finding of sexual dimorphism in LDL-C response to walnut supplementation needs confirmation. WAHA was conducted in free-living individuals, who chose their daily foods, which may be viewed as desirable because it is closer to real life than the situation in controlled feeding studies.

On the basis of associations ascertained in cohort studies,⁵ the observed shift of the lipoprotein subclass phenotype suggests a reduction of lipoprotein-related CVD risk by long-term consumption of walnuts, which provides novel mechanistic insight for their potential cardiovascular benefit beyond effects on the standard lipid panel. Our data reinforce the notion that regular walnut consumption may be a useful part of a multicomponent dietary intervention or dietary pattern to lower atherogenic lipids and improve CVD risk.

Nonstandard Abbreviations and Acronyms

CVD	cardiovascular disease
LDL-C	low-density lipoprotein cholesterol

cardiovascular disease

low-density lipoprotein cholesterol

The authors thank the participants in the trial for their enthusiastic collaboration and E. Corbella for expert statistical assistance. CIBEROBN is an initiative of Instituto de Salud Carlos III, Spain.

This work was supported by a grant from the California Walnut Commission, Folsom, CA. The funding agency had no involvement in the study design, data collection, analyses, and interpretation of the data or writing of the manuscript. Dr Sala-Vila holds a Miguel Servet fellowship (CP17/00029) and is supported by Fondo de Investigación Sanitaria-FEDER (PI15/01014 grant), Instituto de Salud Carlos III, Spain.

Disclosures Drs Rajaram, Sala-Vila, Sabaté, and Ros have received research funding through their institutions from the California Walnut Commission (CWC), Folsom, CA. Dr Sala-Vila has also received support from CWC to attend professional meetings. Drs Sabaté and Ros were nonpaid members of the Scientific Advisory Council of the CWC. Dr Ros was a paid member of the CWC Health Research Advisory Group and has received personal money from the CWC for presentations. All other authors declare no competing interests.

https://www.ahajournals.org/journal/circ

Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01634841.

For Sources of Funding and Disclosures, see page 1085.

中文翻译：

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食用核桃 2 年对健康老年人脂蛋白亚类的影响

经常食用坚果是植物性饮食的重要组成部分，与总心血管疾病 (CVD) 和 CVD 死亡率降低 15% 和 23% 相关。¹小型、短期随机对照试验表明，添加坚果的饮食具有一致的降低胆固醇的作用；然而，没有针对老年人、从不同地理位置招募参与者或持续 2 年的富含坚果的饮食来改变脂质的试验。²此外，关于坚果对脂蛋白亚类的影响的信息也很少。

我们假设将核桃纳入日常饮食会改善血脂状况，而不管地理和饮食背景的差异。WAHA 研究（核桃与健康老龄化）是一项 2 中心（西班牙巴塞罗那和美国加利福尼亚）、为期 2 年的平行组随机对照试验，旨在测试补充核桃饮食对健康老年人的影响（网址：https ://www.clinicaltrials.gov；唯一标识符：NCT01634841）。³脂蛋白变化是预先指定的次要结果。该研究得到了各中心伦理委员会的批准。由于伦理委员会的限制，数据、分析方法和研究材料将不会提供给其他研究人员。符合条件的候选人是认知健康的老年人（63-79 岁），没有严重的合并症。³在提供知情同意后，参与者 (n=708) 被分配到无核桃（对照）或添加核桃的饮食（≈15% 的能量，30-60 g/d）。在 2 个月的访问中，记录依从性、耐受性、药物变化和体重。每次访问时，都会将 8 周的生核桃片分配给相应的组。在整个研究过程中，参与者由他们的主治医师监督，他们根据他们对风险因素水平的评估更换药物，包括降脂药物。

基线和 2 年空腹血糖、胆固醇、甘油三酯和高密度脂蛋白胆固醇通过标准酶法测定；低密度脂蛋白胆固醇 (LDL-C) 由 Friedewald 公式计算。在 Biosfer-Teslab（西班牙雷乌斯）使用 Liposcale 进行高级脂蛋白测试，这是一种经过验证的二维¹ H 核磁共振波谱。⁴我们通过单向方差分析分析了营养摄入和体重的 2 年差异，并通过多变量调整的 ANCOVA 分析了葡萄糖和脂蛋白的变化。

结果显示，636 名参与者完成了研究（90% 的保留率），628 名拥有完整的脂蛋白分析数据（平均年龄 69 岁，67% 女性，32% 接受他汀类药物治疗）。对于主要认知结果，他们的临床特征与完成者的临床特征没有区别。³平均基线 LDL-C 和甘油三酯分别为 117 和 105 mg/dL。不同治疗组的试验中他汀类药物变化没有差异。核桃饮食依从性良好，体重稳定，核桃饮食的平均 2 年变化为 0.06 kg（95% CI，–0.32 至 0.44）和 –0.51 kg（95% CI，–0.91 至 –0.12） ) 在控件中。反映核桃的营养成分，核桃组的参与者增加了能量、总脂肪、纤维、亚油酸和α-亚麻酸的摄入量（图A）。未观察到空腹血糖的组间显着变化（图 B）。核桃饮食显着降低 (mg/dL) 总胆固醇（平均 –8.5 [95% CI，–11.2 至 –5.4]）、LDL-C（平均 –4.3 [–6.6 至 –1.6]）和中密度脂蛋白胆固醇（–1.3 [–1.5 到 –1.0]），对应减少 4.4%、3.6% 和 16.8%，分别，而甘油三酯和高密度脂蛋白胆固醇不受影响（图 B 和 C）。总 LDL 颗粒和小 LDL 颗粒数量分别减少了 4.3% 和 6.1%（图 D）。研究地点的结果没有差异。对核桃饮食的脂质反应因性别而异：男性的 LDL-C 降低了 7.9%，女性降低了 2.6%。P-交互作用=0.007）。

数字。 按干预组划分的饮食变量以及脂质和脂蛋白亚类在 2 年内的平均百分比变化。A、能量和营养摄入。B，测量的脂质和脂蛋白胆固醇浓度。C，通过核磁共振波谱估计的脂蛋白脂质浓度。D, 通过核磁共振波谱估计的脂蛋白颗粒数。误差棒代表 95% CI。ALA表示α-亚麻酸；C、胆固醇；DHA，二十二碳六烯酸；恩，能量；EPA，二十碳五烯酸；HDL，高密度脂蛋白；LA，亚油酸；低密度脂蛋白，低密度脂蛋白；MUFA，单不饱和脂肪酸；P，粒子数；PUFA，多不饱和脂肪酸；SFA，饱和脂肪酸；TG，甘油三酯；和极低密度脂蛋白，极低密度脂蛋白。* P <0.05。通过单向方差分析获得了营养摄入差异的P值。根据中心、年龄、性别、体重指数、吸烟状况（曾经吸烟者/从不吸烟者）、APOE调整的 ANCOVA 获得的脂质和脂蛋白差异的P值ε4 携带（是/否）、体力活动变化、糖尿病（是/否）、血脂异常（是/否）、高血压（是/否）、他汀类药物治疗（是/否）、标准化为辛伐他汀的他汀类药物剂量的变化，以及每个变量的基线值。变化的估计边际平均值用于计算两组相对于基线的百分比变化。

这项为期 2 年的随机对照试验的结果表明，将每日剂量的核桃（约 15% 的能量）与具有基本正常血脂特征的自由生活老年人的习惯性饮食相结合，导致平均 4.3 mg/dL 的 LDL- C 降低，这是适度的，尽管在高胆固醇血症的个体中观察到了更大的反应。²我们的数据还支持核桃饮食对核磁共振评估的脂蛋白亚组分的有益影响，降低中密度脂蛋白胆固醇（残留胆固醇的一个重要因素）和总 LDL 颗粒。前瞻性研究报告称，在 CVD 风险预测中，LDL 颗粒数量始终优于 LDL-C，并且残余胆固醇与 CVD 有因果关系，与 LDL-C 无关。⁵摄入不同饮食的 2 个队列中的脂质反应没有不同，这加强了我们结果的概括。WAHA 是迄今为止规模最大、时间最长的坚果试验，克服了先前较小和较短坚果研究的局限性。对核桃补充剂的 LDL-C 反应中性别二态性的新发现需要证实。WAHA 是在自由生活的个体中进行的，他们选择自己的日常食物，这可能被认为是可取的，因为它比控制喂养研究中的情况更接近现实生活。

根据队列研究中确定的关联，⁵观察到的脂蛋白亚类表型的变化表明长期食用核桃可降低脂蛋白相关的 CVD 风险，这为它们潜在的心血管益处提供了新的机制见解，超越了对心血管的影响。标准脂质面板。我们的数据强化了这样一种观点，即定期食用核桃可能是多组分饮食干预或饮食模式的有用部分，可降低致动脉粥样硬化的脂质和改善 CVD 风险。

非标准缩写和首字母缩略词

化学气相沉积	心血管疾病
低密度脂蛋白胆固醇	低密度脂蛋白胆固醇

心血管疾病

低密度脂蛋白胆固醇

作者感谢试验参与者的热情合作和 E. Corbella 的专家统计协助。CIBEROBN 是西班牙卡洛斯三世研究所的一项倡议。

这项工作得到了加利福尼亚州福尔瑟姆加州核桃委员会的资助。资助机构不参与研究设计、数据收集、分析和数据解释或手稿的写作。Sala-Vila 博士拥有 Miguel Servet 奖学金 (CP17/00029)，并得到西班牙萨鲁德卡洛斯三世研究所 Fondo de Investigación Sanitaria-FEDER（PI15/01014 资助）的支持。

披露Rajaram、Sala-Vila、Sabaté 和 Ros 博士通过他们的机构从加利福尼亚州福尔瑟姆的加州核桃委员会 (CWC) 获得了研究资金。Sala-Vila 博士还得到了 CWC 的支持，可以参加专业会议。Sabaté 和 Ros 博士是 CWC 科学咨询委员会的无薪成员。Ros 博士是 CWC 健康研究咨询小组的付费成员，并从 CWC 收到个人资金用于演讲。所有其他作者声明没有竞争利益。

https://www.ahajournals.org/journal/circ

注册：网址：https://www.clinicaltrials.gov；唯一标识符：NCT01634841。

有关资金来源和披露信息，请参见第 1085 页。