Effect of low carbohydrate high fat diet on LDL cholesterol and gene expression in normal-weight, young adults: A randomized controlled study

TL;DR: The effects of a diet with less than 20 g carbohydrates per day (LCHF) on plasma low density lipoprotein cholesterol (LDL-C) in young and healthy adults and the assessment of lipid profile and peripheral blood mononuclear cells (PBMC) gene expression are explored.

About: This article is published in Atherosclerosis.The article was published on 2018-12-01 and is currently open access. It has received 62 citations till now. The article focuses on the topics: Atkins diet.

Summary

1. Introduction

• The role of low carbohydrate high fat (LCHF) diets in relation to health effects is debated in the scientific literature [1, 2] as well as the role of saturated fatty acids for the development of atherosclerosis despite the overwhelming evidence linking saturated fat to increased LDL-C level [3-5].
• Butter and cream are typically promoted as healthy in LCHF literature, and in Norway, the use of butter increased by 24% during the period 2009-2012 [13].
• Indeed, LCHF diets are used not only by overweight, but by healthy normal-weight people as well [27].

Study participants

• Participants were mostly recruited among students or employees at the Department of Nutrition Research at the University of Oslo after information meetings.
• The participants were included if they were willing to change their dietary intake according to the restrictions in a LCHF diet, provided informed consent and if they fulfilled the inclusion criteria after a physical examination by the study physician.
• Inclusion criteria were age 18 years or older, no chronic disease present, alcohol consumption less than 14 units per week and not following any particular dietary restrictions.
• Exclusion criteria were chronic diseases, regular use of medications (except oral contraceptives), blood pressure ≥140/90 mm Hg, pregnant or lactating women.

Study design and diet

• The study was a three week randomized controlled parallel-designed intervention study, performed in the period September 2011 to December 2012.
• The diet was self-selected, thus the type of fat was optional.
• A partial limited intake existed for some food items such as whole-fat cheeses containing carbohydrates and vegetables with low carbohydrate content, such as spinach, sprouts, mushrooms, cucumber and avocado.
• The study was registered in ClinicalTrials.gov with the ID number: NCT01476436.
• A weighed dietary record was also performed for three days during the LCHF diet period.

Anthropometric measures

• Body weight, fat free mass, fat mass and total body water were determined using a bioimpedance analyzer (Tanita TBF-300, Tanita Corp., Japan) at baseline and after three weeks of LCHF diet.
• The measurement was undertaken according to the manual and the subjects fasted for at least eight hours prior to study visits.
• During the measurement the subjects were in standing position with the arms and legs abducted from the body and instructed not to move or speak.

Blood pressure

• Fasting systolic and diastolic blood pressure (BP) measurements were performed by trained personnel.
• Three measurements at 1-minute intervals were recorded after 10 minutes of rest in a waiting room followed by another 5 minutes in an investigation room where the subject sat in a resting chair with the cuff mounted and the arm at the armrest.
• Validated oscillometric devices (Carescape V100, GE Healthcare, Norway) with suitable cuffs were used for the measurements.
• In the analyses the authors used the mean of all three measurements.

Routine laboratory analysis

• Fasting blood samples were drawn at baseline and after three weeks.
• Plasma was obtained from EDTA tubes (Becton Dickinson), immediately placed on ice and centrifuged within 10 minutes (1500 rpm, 4°C, 15 minutes).
• Plasma (P)-TC and P-triglyceride (TG) was measured with an enzymatic colorimetric assay, while P-LDL-C and P-high density lipoprotein cholesterol (HDL-C) was measured with a homogeneous enzymatic colorimetric assay.
• Four hundred ng of RNA from all samples was reverse transcribed using High capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA).
• Ct values of the two reference genes (=ΔCt).

Statistical analyses

• Data are presented as mean ± standard deviation (SD) if normally distributed, or median (minimum-maximum) if not normally distributed.
• Wilcoxon signed rank test was used to assess change within groups and Mann Whitney U test was used to compare changes between groups when the data was not normally distributed.
• IBM SPSS Statistics v 20 (Armonk, NY, USA) was used for statistical analysis.
• In the present study the authors used a conservative approach and hypothesized that Atkins diet would lead to an increase in LDL-C from 2.0 to 2.5 mmol/l, which is a less increase than what they observed in a small pilot study [33].
• With 80% power and a level of significance of 0.05 and a SD of 0.5 mmol for LDL-C the necessary sample size (for each sample separately) was 16.

3. Results

• Forty-one subjects met to the screening visit and two did not fulfill the inclusion criteria.
• Table 1 shows the baseline and end of study characteristics of the participants.
• The routine laboratory measurements for lipids, glucose and CRP were within the reference value in all participants at baseline (Table 1).
• During the study period, weight and BMI was significantly reduced in both the LCHF and the control group (1.2 kg and 2.0 kg and 0.4 kg/m2 and 0.7 kg/m2, respectively).

Dietary intake and compliance

• A total of 18 participants performed a weighed dietary record of their habitual diet and 10 participants performed a weighed dietary record of their LCHF diet (Table 2).
• The E% from total fat, saturated fat, monounsaturated fat, polyunsaturated fat, protein, carbohydrate and dietary cholesterol differed significantly between the habitual diet and the LCHF diet (all p<0.001).
• Expression of LDL receptor and other lipid-related genes and circulating PCSK9 level.
• One of the suggested mechanisms by which saturated fat increase LDL-C levels is through modulation of the LDL receptor.
• The gene expression of the transcription factor SREBP-1 were significantly different between the LCHF and control group after three weeks (p=0.01) (Table 4), whereas there were no significant difference between the groups in the change of the expression of the other lipidrelated genes.

Adverse events

• There were two serious adverse events during the study, both occurred in the LCHF group.
• One healthy participant experienced chest pain three days after starting on the LCHF diet.
• ST-elevation was observed at the electro cardiogram at admission to hospital and severely elevated serum levels of Troponins and Creatine kinase-MB were observed.
• The patient was excluded from the study and partly recovered after standard medical treatment and restarting eating carbohydrate.

4. Discussion

• An important finding of this study is, that after three weeks on a LCHF diet, mean plasma LDL-C increased by 44% compared to the control group.
• An interesting finding in the present study was the striking differences between individuals in the response to a LCHF diet, in particular for plasma levels of LDL-C.
• In the present study, the authors observed a 30%, but not statistically significant decrease, in LDL receptor gene expression in the LCHF group.
• Other study limitations are the small study population albeit the p-values on primary end points were strong.
• The metabolic stress caused by this diet may be used as a model for research on the regulation of LDL-C levels in human since little is known about the molecular mechanisms behind the large individual differences.

Conflict of interest

• K.R. has received grants or honoraria for meeting and lectures the last three years from Amgen, Chiesi, Sanofi, Mills DA, MSD , Oslo Economics, Takeda outside the submitted work and has received honoraria for participation in meetings for Norwegian Directorate of Health and the Norwegian Medical Association outside the submitted work.
• K.B.H has received grants from TINE SA, Mills DA, Olympic Seafood, Amgen, Sanofi, Kaneka, and Pronova outside the submitted work.

Financial support

• The authors thank The Mills Vita Heart Foundation, The Throne Holst Foundation, Aktieselskabet Freia Chocolade Fabriks Medisinske Fond, Norsk Fond for Klinisk Kjemi, Dr. Fürst medisinske laboratoriums fond til klinisk kjemisk og klinisk fysiologisk forskning for financial support to this study.
• The funding bodies had no role in the conduct of the research and/or preparation of the article, study design, collection, analysis or interpretation of data, neither in the writing of the report; or in the decision to submit the article for publication.

Author contributions

• K.B.H., M.S. and K.R conceived of the idea and planned the experiments.
• K.R. conducted the clinical part of the study, K.B.H. conducted the biochemical and genetic analysis and was responsible for the statistical work, I.N. conducted the genetic analysis and M.S conducted the nutritional measurement and.
• All authors did statistical work, discussed and interpreted the results and made substantial contributions to the final manuscript.

Figure Legends

• R denotes randomization, Habitual diet denotes the control arm, LCHF diet denotes low carbohydrate high fat diet.
• Each bar represent one subject’s percent change in plasma lipids, as indicated by the text in each panel A-F.
• The fasting plasma levels at baseline were compared to fasting plasma levels after using LCHF for 3 weeks expressed as percent change.
• Subjects were ranked in order after the percent increase in LDL-C, from least change to the left side in the panel and the largest change to the right side of the panel.
• Panel A: LDL-C; Low density lipoprotein cholesterol, Panel B: TC; Total cholesterol, Panel C: HDL-C;.

Figures

Table 2. Dietary intake on habitual diet and on LCHF diet.

Table 4. Expression of lipid-related genes in PBMC in the LCHF and control group

Table 3. List of lipid-related genes analyzed in peripheral blood mononuclear cells before and after LCHF intervention

Frequently asked questions

Q1. What was used to assess change between groups when the data was not normally distributed?

Wilcoxon signed rank test was used to assess change within groups and Mann Whitney U test was used to compare changes between groups when the data was not normally distributed. 

Q2. What contributions have the authors mentioned in the paper "Effect of low carbohydrate high fat diet on ldl cholesterol and gene expression in normal-weight, young adults: a randomized controlled study" ?

The role of low carbohydrate high fat ( LCHF ) diets in relation to health effects is debated in the scientific literature this paper. 

Q3. What are the future works in "Effect of low carbohydrate high fat diet on ldl cholesterol and gene expression in normal-weight, young adults: a randomized controlled study" ?

Taken together, further research is needed on how this class of drugs affects lipid metabolism. In clinical practice, the unpredictable response to the LCHF diet suggests that LDL-C should be measured in people using a LCHF diet. 

Q4. How much carbohydrate is allowed in the Atkin diet?

Less than 20 grams carbohydrates per day are allowed in the initially phase of the Atkin diet, typically resulting in a high intake of saturated fat. 

Q5. What was the subject's position during the measurement?

During the measurement the subjects were in standing position with the arms and legs abducted from the body and instructed not to move or speak. 

Q6. How many people have been on a low carbohydrate diet in the last 12 months?

Population surveys in Norway have reported that 29% of adult women and 21% of men had been on a “low carbohydrate diet” the last 12 months [27]. 

Q7. Why did the LCHF group have a restriction on alcohol intake?

Due to diet restrictions on carbohydrates, beer, wine and liquor containing carbohydrate alcohol was naturally limited in the LCHF group but there were no restriction on alcohol intake as such. 

Q8. How many people reported that the Atkins diet helped them to lose weight?

In a study of more than 32,000 U.S. dieters, nearly 34% of respondents reported that the Atkins diet helped them to loose and maintain weight [10]. 

Q9. What are the key regulators of the LDL-C level?

Together with proprotein convertase subtilisin/kexin type 9 (PCSK9), which modulate the degradation of LDL receptors [30], these factors are key regulators of the serum LDL-C level. 

Q10. What is the role of LCHF diet in cardiovascular risk factors?

It is important to gain knowledge of how LCHF diet influence cardiovascular risk factors in normal-weight subjects since the use of LCHF diets is popular and not restricted to weight reduction in obesity only. 

Q11. How many participants were included in the meta-analysis?

Another meta-analysis of eleven RCT’s with 1369 participants using a diet with less than 20 E% carbohydrate also observed an increase in LDL-C (0.16 mmol/l, 95% CI: 0.003, 0.33) [22]. 

Q12. What was the effect of the Atkins diet on LDL-C?

In the present study the authors used a conservative approach and hypothesized that Atkins diet would lead to an increase in LDL-C from 2.0 to 2.5 mmol/l, which is a less increase than what the authors observed in a small pilot study [33]. 

Q13. How did the participants respond to the LCHF diet?

The participants were included if they were willing to change their dietary intake according to the restrictions in a LCHF diet, provided informed consent and if they fulfilled the inclusion criteria after a physical examination by the study physician. 

Q14. What is the role of low carbohydrate diets in relation to health effects?

A meta-analysis on the effect of low carbohydrate diets with 23 RCT`s reported a statistically significant higher LDL-C (0.1 mmol/l, 95% CI: 0.026- 0.165) in the low-carbohydrate versus the low-fat diet groups [21]. 

Q15. What was the RNA quantity and quality of the PBMCs?

All analyzes were accredited after International and European standard NS-EN ISO 15189Expression of lipid-related genes in peripheral blood mononuclear cells (PBMCs)After blood collection, PBMCs were isolated by using the BD Vacutainer CellPreparation tubes with sodium heparin according to the manufacturer (Becton Dickinson, San Jose, CA) and stored as pellets at -80o C until further mRNA isolation. 

Q16. What was the RNA quantity and quality measured?

RNA quantity and quality measurements were performedusing ND 1000 Spectrophotometer (Saveen Werner Carlson Circle Tampa, FL) and Agilent Bioanalyser (Agilent Technology, Santa Clara, CA), respectively.