Postprandial Apolipoprotein B48 is Associated with Subclinical Atherosclerosis in Patients with Rheumatoid Arthritis

Round 1

Reviewer 1 Report

The concept studied, postprandial lipidemia and apoB-impact is interesting and original, not so much has been done in this field in rheumatology and RA. 

The age of the patients and controls has to be clarified, is it 59 (patients) or 56 as is stated somewhere. If there is about 10% age difference between case and controls which is written in table 1, this could have big impact on conclusions since postprandial effects usually are associated with age. I guess there is an error in the. table, but this must be clarified. 

The association reported is weak, borderline significant actually, and the discussion should be a little more clear about this. 

Author Response

Reviewer #1: 

The concept studied, postprandial lipidemia and apoB-impact is interesting and original, not so much has been done in this field in rheumatology and RA. 

The age of the patients and controls has to be clarified, is it 59 (patients) or 56 as is stated somewhere. If there is about 10% age difference between case and controls which is written in table 1, this could have big impact on conclusions since postprandial effects usually are associated with age. I guess there is an error in the. table, but this must be clarified. 

R:  Thanks for the comment. It is certainly a digitization error in the table. The correct age of the cases is 55.8 (11.4) as it is correctly written into the text. We revised the rest of the table in order to find any other error but everything else is ok. Finally, this variable has been expressed as median at the request of another reviewer:

Table 1. Baseline characteristics of 40 patients with RA and 40 controls.

Variable	Patients n=40	Controls n=40	p Value
Epidemiological characteristics
Age in years, median (IQR)	55.8 (52.9-61.7)	57.0 (52.7-61.1)	0.662

 

The association reported is weak, borderline significant actually, and the discussion should be a little clearer about this. 

R: We agree with your comment, and it is probably due to our sample size.  We added this limitation to the discussion.

Line 387-389: “Finally, although differences in postprandial ApoB48 between patients and controls were significative according to the design of our study, the weak significance of the results should be taken into account.”

Author Response File: Author Response.pdf

Reviewer 2 Report

The work of Mena-Vázquez N. et al analyzed cardiovascular risk factors in RA patients and they found that the apolipoprotein B48 is associated with the cIMT. The work is properly designed and the results clearly presented. However I have some concerns related with the interpretation of the results and I miss some analysis.

Major points

1. The authors found differences in the levels of blood lipids and ApoB48 between RA patients with pathologic cIMT and patients with normal pathologic cIMT. But are these differences also found in the control group? This is an important point because it will determine if these differences are specific of the disease.

2. Related to this, do the authors find these differences if they exclude the men from the comparations? Because as they explain in the discussion the gender has a great influence in the cardiovascular risk and therefore the higher risk may be only due to a higher frequency of men in the pathologic cIMT group.

3. The authors should also discuss in more detail the effect of glucocorticoids, as the use of them has been associated with a higher cardiovascular risk in RA patients (Roubille C, Richer V, Starnino T, et al The effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis. Annals of the Rheumatic Diseases 2015;74:480-489).

Minor points

1. ACPAs levels were higher in patients with pathologic cIMT (p=0.036). I recommend showing the correlation analysis.

2. In table 1 the VLDL are exactly the same in both control and RA groups in the fasting and postprandial. Is that correct or there is an error in the table?

3. In all the tables the authors should include the IQR instead the SD.

4. Why secondary Sjögren syndrome was not an exclusion criteria?

    

Author Response

Reviewer #2: 

Major points

1. The authors found differences in the levels of blood lipids and ApoB48 between RA patients with pathologic cIMT and patients with normal pathologic cIMT. But are these differences also found in the control group? This is an important point because it will determine if these differences are specific of the disease.

R:  These differences were not found in the levels of blood lipids and ApoB48 between control group with pathologic cIMT and control group with normal cIMT.  The results in the control group are shown in the following table (we added this table in supplementary material).

We have added in results, line 254-256: These differences were not found in the levels of blood lipids and ApoB48 between control group with pathologic cIMT and control group with normal cIMT (supplementary material).

We have added in the discussion, line 341-344: “The differences found in apob48 between individuals with pathological and non-pathological cIMT were only found in the patient group and not in the control group, so this difference may be more related to the disease itself and not to other frequently associated factors with the lipids level”.

 

Supplementary Table 1. Lipid profile and carotid ultrasound in control group according to cIMT.

Variable	Control group with IMT >p90 n=9		Control group with IMT ≤p90 n=31			Control group with IMT >p90 vs with IMT ≤p90 p-value
	Fasting	Postprandial	Fasting		Postprandial	Fasting	Postprandial
Fasting lipid profile
Total cholesterol (mg/dl), median (IQR)	190.0 (171.1-226.5)	189.0 (166.5-220.5)	202.0 (176.0-236.0)	201.5 (167.7-227.0)		0.656	0.678
LDL cholesterol (mg/dl), median (IQR)	110.0 (83.5-148.0)	108.0 (75.5-131.5)	120.0 (96.0-142.0)	108.5 (83.0-128.0)		0.667	0.638
HDL cholesterol (mg/dl), median (IQR)	56.0 (45.5-74.0)	53.0 (46.5-68.5)	60.0 (48.0-71.0)	58.0 (45.0-66.0)		0.762	0.830
Triglycerides (mg/dl), median (IQR)	72.0 (54.5-140.5)	116.5 (67.7-206.7)	86.0 (60.0-128.0)	136.0 (97.2-170.0)		0.545	0.473
Chylomicrons (triglycerides), median (IQR)	15.2 (5.3-42.9)	51.2 (15.3-78.2)	17.0 (7.5-45.3)	54.3 (32.4-80.7)		0.831	0.473
Chylomicrons (cholesterol), median (IQR)	5.5 (2.6-22.4)	29.1 (5.5-36.8)	11.0 (7.5-45.3)	14.1 (8.7-32.9)		0.509	0.497
VLDL (triglycerides), median (IQR)	13.6 (8.5-43.7)	24.2 (11.2-57.1)	20.7 (9.8-31.1)	25.0 (17.9-38.1)		0.935	0.911
VLDL (cholesterol), median (IQR)	5.9 (1.6-14.1)	7.1 (4.0-15.6)	5.7 (2.8-8.3)	7.4 (3.9-10.6)		0.730	0.543
ApoB48, median (IQR)	5.2 (4.6-8.0)	14.4 (9.4-15.6)	8.3 (6.4-11.8)	13.9 (11.7-17.2)		0.116	0.274
ApoB total, median (IQR)	101.9 (80.0-108.0)	94.1 (74.0-105.1)	96.8 (82.4-111.2	92.9 (76.7-104.0)		0.975	0.846
Increased postprandial blood lipids
Triglycerides (mg/dl), median (IQR)		42.7 (16.1-128.8)		48.4 (32.2-75.8)			0.709
Chylomicrons (triglycerides), median (IQR)		29.3 (11.8-135.3)		33.5 (13.1-64.0)			0.289
VLDL (triglycerides), median (IQR)		9.8 (2.2-22.4)		8.4 (2.0-17.7)			0.975
ApoB48, median (IQR)		6.7 (3.3-9.9)		6.9 (2.2-9.8)			0.920

 

* p <0.005 fasting vs postprandial value. Abbreviations: cIMT, carotid intima media thickness; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; VLDL, very-low-density lipoprotein.

2. Related to this, do the authors find these differences if they exclude the men from the comparations? Because as they explain in the discussion the gender has a great influence in the cardiovascular risk and therefore the higher risk may be only due to a higher frequency of men in the pathologic cIMT group.

R:  The results obtained are similar when we excluded men from the comparations, except for the HDL values. The patients with pathologic cIMT did not have higher levels of HDL cholesterol. However, an increase in lipids and ApoB48 was observed between RA patients with pathologic cIMT and patients with normal pathologic cIMT. 

With respect to pre-prandial blood lipids, patients with pathologic cIMT had higher levels of triglycerides (p=0.021) and higher levels of VLDL (triglycerides) (p=0.011), and Chylomicrons (triglycerides)(p=0.011) than the patients with normal cIMT. Similar results were recorded for postprandial blood lipids, with higher levels of triglycerides (p=0.042), chylomicrons (triglycerides) (p=0.052), and VLDL (cholesterol) (p=0.048) in patients with pathologic cIMT. There were no preprandial differences in apolipoproteins, although postprandial ApoB48 was higher in patients with pathologic cIMT (p=0.046). Furthermore, after a mixed breakfast, the increase in ApoB48 and chylomicrons (triglyceride) was significantly greater in patients with pathologic cIMT (p=0.032 and p=0.042, respectively). These results are shown in the following table for the reviewer.

We added in the text line 338-341: Similarly, we did not find an association between HDL and pathologic cIMT, since HDL was eliminated from the multivariate model when we included sex, owing to the weight of this factor and to the higher number of men with pathologic cIMT (supplementary material).

We added the next table to supplementary material:

Supplementary Table 2. Lipid profile and carotid ultrasound in RA patients according to cIMT excluded the men.

 

Variable	RA with IMT >p90 n=6		RA with IMT ≤p90 n=29		RA with IMT >p90 vs RA with IMT ≤p90 p
	Fasting	Postprandial	Fasting	Postprandial	Fasting	Postprandial
Fasting lipid profile
Total cholesterol (mg/dl), median (IQR)	234.5 (212.7-255.7)	210.0 (195.2-252.5)	212.0 (185.5-227.5)	200.0 (177.0-228.0)	0.176	0.235
LDL cholesterol (mg/dl), median (IQR)	139.0 (133.0-165.5)	116.0 (105.7-145.0)	122.0 (103.5-140.5)	110.0 (94.0-129.5)	0.164	0.312
HDL cholesterol (mg/dl), median (IQR)	66.5 (60.2-78.2)	62.5 (55.7-72.2)	66.0 (53.5-80.5)	62.0 (52.5-77.0)	0.749	0.783
Triglycerides (mg/dl), median (IQR)	112.0 (83.5-165.7)	195.0 (125.2-326.5)	74.0 (63.0-105.0)	111.0 (83.0-163.5)	0.021	0.042
Chylomicrons (triglycerides), median (IQR)	52.7 (16.6-64.9) *	77.7 (22.9-195.4) *	13.8 (8.7-20.9)	34.2 (21.7-48.9)	0.011	0.052
Chylomicrons (cholesterol), median (IQR)	13.4 (8.0-23.3)	15.5 (5.7-33.2)	8.5 (6.0-10.7)	14.3 (7.2-23.2)	0.093	0.782
VLDL (triglycerides), median (IQR)	27.2 (21.9-42.4)	37.3 (17.3-60.0)	13.8 (9.7-23.1)	20.7 (12.9-38.7)	0.011	0.098
VLDL (cholesterol), median (IQR)	5.9 (4.9-10.7)	8.8 (3.4-12.7)	2.7 (1.9-5.4)	3.9 (2.6-8.9)	0.014	0.048
ApoB48, median (IQR)	7.5 (5.5-12.1) *	23.7 (10.0-31.1) *	7.0 (6.1-10.2)	13.4 (10.4-18.4)	0.685	0.046
ApoB total, median (IQR)	104.0 (98.9-120.8)	106.3 (89.8-114.0)	94.5 (79.2-105.5)	91.9 (75.3-103.0)	0.093	0.062
Increased postprandial blood lipids
Triglycerides (mg/dl), median (IQR)		63.7 (16.2-140.0)		35.7 (17.1-61.6)		0.279
Chylomicrons (triglycerides), median (IQR)		46.4 (11.0-147.0)		20.0 (10.2-34.7)		0.042
VLDL (triglycerides), median (IQR)		11.8 (6.4-22.0)		7.0 (3.0-16.3)		0.454
ApoB48, median (IQR)		13.0 (6.2-16.4)		6.7 (3.4-8.4)		0.032

 

* p <0.005 fasting vs postprandial value. Abbreviations: cIMT, carotid intima media thickness; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; VLDL, very-low-density lipoprotein.

3. The authors should also discuss in more detail the effect of glucocorticoids, as the use of them has been associated with a higher cardiovascular risk in RA patients (Roubille C, Richer V, Starnino T, et al The effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis. Annals of the Rheumatic Diseases 2015;74:480-489).

R: We have included the glucocorticosteroids variable in multivariate logistic regression analysis (dependent variable [DV]: pathologic cIMT) and similarly in alternative multivariate linear regression analysis (DV: cIMT). We have changed table 5 and table 6. However, this study was not designed to assess the effect of drugs, including glucocorticoids, on cIMT

Table 5. Logistic regression of characteristics associated with pathologic cIMT (p>90) in patients with rheumatoid arthritis.

Predictor	OR	95% CI	p Value
Female sex	0.010	0.000 – 0.381	0.014
Postprandial ApoB48*	1.159	1.021- 1.315	0.023
Total ApoB	1.121	1.109-1.259	0.046

Nagelkerke R² = 0.450, Variables not included in the equation: age, HDL post, TG post, ACPA >340, Framingham, smoking, glucocorticosteroids. *ApoB48 log transformed.

Table 6. Multiple linear regression of characteristics associated with cIMT in patients with RA.

Dependent variable	Predictor	B	95% CI for B	p Value
Pathologic cIMT	Female sex	–0.607	–0.306 to –0.151	<0.001
	Postprandial ApoB48	0.285	0.002 to 0.013	0.002
	Total ApoB	0.239	0.001 to 0.005	0.047
	ACPA≥340	0.256	0.018 to 0.137	0.018

Nagelkerke R² = 0,520, Variables not included in the equation: age, HDL post, TG post, Framingham, smoking, and glucocorticosteroids.

 

 

We added in the text, line 383-387:  Corticosteroids treatment was higher in patients with pathologic cIMT. However, this variable was eliminated from the multivariate model, probably due to the weight of lipid effect. In this sense, some authors have reported a relationship between glucocorticosteroids and cardiovascular events by increasing the risk due to deleterious effects on lipids, among other causes (1, 2).

 

We added references, line 554-560:

1. Roubille C, Richer V, Starnino T, McCourt C, McFarlane A, Fleming P, et al. The effects of tumour necrosis factor inhibitors, methotrexate, non-steroidal anti-inflammatory drugs and corticosteroids on cardiovascular events in rheumatoid arthritis, psoriasis and psoriatic arthritis: a systematic review and meta-analysis. Ann Rheum Dis. 2015;74(3):480-9.
2. Panoulas VF, Douglas KM, Stavropoulos-Kalinoglou A, Metsios GS, Nightingale P, Kita MD, et al. Long-term exposure to medium-dose glucocorticoid therapy associates with hypertension in patients with rheumatoid arthritis. Rheumatology (Oxford). 2008;47(1):72-5.

 

Minor points

1. ACPAs levels were higher in patients with pathologic cIMT (p=0.036). I recommend showing the correlation analysis.

R: When analyzing the ACPA levels with the maximum GIMc as a quantitative variable, the correlation analysis did not show significant differences although it shows a positive correlation with a tend statistical significance (r= 0.290; p= 0.096).

 

We added in the text, line 223-227: There were no differences in the clinical and laboratory data, except that ACPAs were more frequently positive and with higher titters (>340) in patients with pathologic cIMT (p=0.036). The correlation analysis did not show significant differences although it shows a positive correlation with a tend statistical significance (r= 0.290; p= 0.096) (Figure 1). There were no differences in DMARDs, although patients with pathologic cIMT more frequently took glucocorticoids (p=0.032).

 

We added Figure 1:

 

2. In table 1 the VLDL are exactly the same in both control and RA groups in the fasting and postprandial. Is that correct or there is an error in the table?

R: Thanks for the comment. It is certainly a digitization error in the table.  We have changed in table 2 median and p-value.

Table 2. Lipid profile and carotid ultrasound in 40 patients with RA and 40 controls.

Variable	RA n=40		Controls n=40		RA vs Controls p
	Fasting	Postprandial	Fasting	Postprandial	Fasting	Postprandial
Fasting lipid profile
Total cholesterol (mg/dl), median (IQR)	212.1 (187.0-234.2)	202.0 (178.0-226.2)	200.2 (176.0-227.2)	201.0 (168.1-220.5)	0.148	0.222
LDL cholesterol (mg/dl), median (IQR)	127.0 (107.1-140.0)	110.3 (98.5-130.0)	116.5 (95.7-140.5)	108.0 (83.1-128.6)	0.229	0.203
HDL cholesterol (mg/dl), median (IQR)	62.5 (54.7-76.5)	62.2 (52.7-72.2)	59.5 (47.7-71)	57.1 (46.2-67.2)	0.162	0.063
Triglycerides (mg/dl), median (IQR)	82.5 (66.7-113.5)	130.0 (91.7-185.0) *	88.5 (64.5-125.7)	132.5 (108.2-210.4) *	0.823	0.913
Chylomicrons (triglycerides), median (IQR)	14.7 (10.3-27.4)	42.3 (22.1-81.3) *	16.4 (7.5-39.8)	43.7 (31.9-84.7) *	0.644	0.225
Chylomicrons (cholesterol), median (IQR)	9.2 (6.8-13.5)	9.2 (6.8-13.5) *	12.3 (5.3-21.9)	12.3 (5.3-21.9) *	0.544	0.613
VLDL (triglycerides), median (IQR)	16.0 (10.1-27.7)	29.6 (15.5-41.1) *	21.0 (9.8-31.5)	24.6 (17.6-38.7) *	0.758	0.859
VLDL (cholesterol), median (IQR)	3.6 (2.3-6.1)	3.6 (2.3-6.1) *	5.8 (2.7-9.8)	5.8 (2.7-9.8) *	0.087	0.083
ApoB48, median (IQR)	7.4 (6.2-10.5)	14.4 (10.8-23.2) *	7.7 (5.5- 10.3)	12.1 (10.9-16.2) *	0.874	0.042
ApoB total, median (IQR)	96.1 (84.9-104.9)	92.4 (80-103.4)	98.0 (84.3-108.3)	93.2 (77.3-102.9)	0.950	0.517
TG/HDL ratio, median (IQR)	1.2 (0.8-2.0)	2.1 (1.3-3.5)	1.4 (0.8-2.9)	2.5 (1.6-4.1)	0.597	0.574
ApoB48/TG ratio, median (IQR)	0.09 (0.07-0.1)	0.1 (0.7-0.1)	0.08 (0.1-0.13)	0.09 (0.7-0.1)	0.985	0.326
Fasting carbohydrate profile
Baseline blood sugar (mg/dl), median (IQR)	78.0 (74.7-83)		80.0 (72.7-88.2)		0.843	0.277
Homocysteine, median (IQR)	14.4 (12.8-18)		13.5 (11.4- 16.8)		0.494
Carotid ultrasound
Pathologic cIMT >p90, n (%)	10 (25.0)		9 (22.5)		0.555
Right cIMT (mm), median (IQR)	0.7 (0.6-0.8)		0.7 (0.7-1.0)		0.652
Left cIMT (mm), median (IQR)	0.66 (0.6-0.7)		0.7 (0.64-0.78)		0.353
Patients with atheromatous plaques, n (%)	7 (18.4)		8 (20.0)		0.481

* p <0.005 fasting vs postprandial value. Abbreviations cIMT, carotid intima media thickness; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; VLDL, very-low-density lipoproteins.

 

 

3. In all the tables the authors should include the IQR instead the SD.

R: Following the reviewer's recommendations, we have changed all the tables with median (IQR) instead the mean (SD).

Table 1. Baseline characteristics of 40 patients with RA and 40 controls.

Variable	Patients n=40	Controls n=40	p Value
Epidemiological characteristics
Age in years, median (IQR)	55.7 (52.9-61.7)	57.0 (52.7-61.1)	0.662
Female sex; n (%)	35 (87.5)	34 (85.0)	0.745
Smoking			0.181
Never smoked, n (%)	15 (37.5)	21 (52.5)
Exsmoker, n (%)	19 (47.5)	11 (27.5)
Active smoker, n (%)	6 (15.0)	8 (20.0)
Comorbidities
Arterial hypertension, n (%)	10 (25.0)	9 (22.5)	0.792
Diabetes mellitus, n (%)	2 (5.0)	4 (10.0)	0.395
Cardiovascular disease, n (%)	3 (7.5)	2 (5.0)	0.644
Family history of coronary artery disease, n (%)	13 (32.5)	7 (17.5)	0.121
Anthropometric characteristics
BMI (kg/m2), median (IQR)	26.7 (24.5-31.0)	27.2 (24.4-30.8)	0.758
Obesity, n (%)	10 (26.3)	10 (27.0)	0.944
Waist circumference, (cm), median (IQR)	91.5 (83.0-108.5)	91 (84.0-102.0)	0.361
Hip circumference (cm), median (IQR)	106.5 (103.5-112.3)	105.0 (100.0-114.0)	0.308
Waist-hip ratio, median (IQR)	0.86 (0.8-0.9)	0.86 (0.8-0.9)	0.828
MET-minute, median (IQR)	495.0 (70.0-990.0)	893.0 (280.5-1188.0)	0.008
Total MEDAS score, median (IQR)	10.0 (8.0-11.0)	9.0 (8.0-11.0)	0.184
Framingham %, median (IQR)	2.6 (0.9-4.1)	1.8 (0.7-4.6)	0.501
High risk, n (%)	0 (0.0)	0 (0.0)	1.000
Intermediate risk, n (%)	6 (16.2)	3 (8.1)	0.286
Low risk, n (%)	31 (83.8)	34 (91.9)	0.286
Clinical-laboratory characteristics
Progression of RA, months, median (IQR)	119 (81.2-167.9)	-	-
Diagnostic delay, months, median (IQR)	8.1 (5.6-16.7)	-	-
Erosions, n (%)	16 (40.0)	-	-
RF >10, n (%)	26 (65.0)	0 (0.0)	<0.001
ACPA >20, n (%)	31 (77.5)	0 (0.0)	<0.001
High-sensitivity CRP (mg/dl), median (IQR)	4.2 (2.7-7.4)	1.7 (0.8-3.1)	0.002
ESR (mm/h), median (IQR)	15 (9.0-26.5)	11 (6.6-18.5)	0.016
DAS28 at protocol, median (IQR)	3.06 (2.5-4.2)	-	-
Remission-low activity, n (%)	21 (53.8)	-	-
Moderate-high activity, n (%)	18 (46.1)	-	-
HAQ, median (IQR)	0.9 (0.2-1.6)	-	-
Synthetic DMARDs, n (%)	31 (77.5)	-	-
Methotrexate, n (%)	23 (62.2)	-	-
Leflunomide, n (%)	3 (8.1)	-	-
Sulfasalazine, n (%)	3 (8.1)	-	-
Hydroxychloroquine, n (%)	2 (5.4)
Biologic DMARDs, n (%)	21 (52.5)	-	-
Anti TNF-α, n (%)	17 (45.9)	-	-
Jak inhibitor, n (%)	1 (2.7)	-	-
Anti-IL-6, n (%)	3 (8.1)	-	-
Glucocorticoid at protocol, n (%)	13 (32.5)	-	-
Glucocorticoid dose at protocol, median (IQR)	5 (5.0-5.0)	-	-
Other treatments
Antihypertensive drugs	10 (25.0)	9 (22.5)	0.792
ACEIs, n (%)	7 (17.5)	7 (17.5)	0.778
ARAIIs, n (%)	3 (7.5)	2 (5.0)	0.462
Diuretics, n (%)	5 (12.5)	8 (20.0)	0.370
Metformin, n (%)	2 (5.0)	3 (7.5)	0.320
Insulin, n (%)	0 (0.0)	1 (2.5)	0.320
Other oral antidiabetic agents, n (%)	0 (0.0)	1 (2.5)	0.320

Abbreviations: RA, rheumatoid arthritis; ACPA, anti-citrullinated peptide antibodies; RF, rheumatoid factor; SD, standard deviation; MEDAS, Mediterranean Diet Adherence Survey; DAS28, 28-joint Disease Activity Score; HAQ, Health Assessment Questionnaire; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; DMARD, disease-modifying antirheumatic drug; IL-6, interleukin 6; Anti TNF, anti–tumor necrosis factor ACEI, angiotensin-converting enzyme inhibitor; ARAII, angiotensin II receptor antagonists.

 

Table 2. Lipid profile and carotid ultrasound in 40 patients with RA and 40 controls.

Variable	RA n=40		Controls n=40		RA vs Controls p
	Fasting	Postprandial	Fasting	Postprandial	Fasting	Postprandial
Fasting lipid profile
Total cholesterol (mg/dl), median (IQR)	212.1 (187.0-234.2)	202.0 (178.0-226.2)	200.2 (176.0-227.2)	201.0 (168.1-220.5)	0.148	0.222
LDL cholesterol (mg/dl), median (IQR)	127.0 (107.1-140.0)	110.3 (98.5-130.0)	116.5 (95.7-140.5)	108.0 (83.1-128.6)	0.229	0.203
HDL cholesterol (mg/dl), median (IQR)	62.5 (54.7-76.5)	62.2 (52.7-72.2)	59.5 (47.7-71)	57.1 (46.2-67.2)	0.162	0.063
Triglycerides (mg/dl), median (IQR)	82.5 (66.7-113.5)	130.0 (91.7-185.0) *	88.5 (64.5-125.7)	132.5 (108.2-210.4) *	0.823	0.913
Chylomicrons (triglycerides), median (IQR)	14.7 (10.3-27.4)	42.3 (22.1-81.3) *	16.4 (7.5-39.8)	43.7 (31.9-84.7) *	0.644	0.225
Chylomicrons (cholesterol), median (IQR)	9.2 (6.8-13.5)	9.2 (6.8-13.5) *	12.3 (5.3-21.9)	12.3 (5.3-21.9) *	0.544	0.613
VLDL (triglycerides), median (IQR)	16.0 (10.1-27.7)	29.6 (15.5-41.1) *	21.0 (9.8-31.5)	24.6 (17.6-38.7) *	0.758	0.859
VLDL (cholesterol), median (IQR)	3.6 (2.3-6.1)	3.6 (2.3-6.1) *	5.8 (2.7-9.8)	5.8 (2.7-9.8) *	0.087	0.083
ApoB48, median (IQR)	7.4 (6.2-10.5)	14.4 (10.8-23.2) *	7.7 (5.5- 10.3)	12.1 (10.9-16.2) *	0.874	0.042
ApoB total, median (IQR)	96.1 (84.9-104.9)	92.4 (80-103.4)	98.0 (84.3-108.3)	93.2 (77.3-102.9)	0.950	0.517
TG/HDL ratio, median (IQR)	1.2 (0.8-2.0)	2.1 (1.3-3.5)	1.4 (0.8-2.9)	2.5 (1.6-4.1)	0.597	0.574
ApoB48/TG ratio, median (IQR)	0.09 (0.07-0.1)	0.1 (0.7-0.1)	0.08 (0.1-0.13)	0.09 (0.7-0.1)	0.985	0.326
Fasting carbohydrate profile
Baseline blood sugar (mg/dl), median (IQR)	78.0 (74.7-83)		80.0 (72.7-88.2)		0.843	0.277
Homocysteine, median (IQR)	14.4 (12.8-18)		13.5 (11.4- 16.8)		0.494
Carotid ultrasound
Pathologic cIMT >p90, n (%)	10 (25.0)		9 (22.5)		0.555
Right cIMT (mm), median (IQR)	0.7 (0.6-0.8)		0.7 (0.7-1.0)		0.652
Left cIMT (mm), median (IQR)	0.66 (0.6-0.7)		0.7 (0.64-0.78)		0.353
Patients with atheromatous plaques, n (%)	7 (18.4)		8 (20.0)		0.481

* p <0.005 fasting vs postprandial value. Abbreviations cIMT, carotid intima media thickness; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; VLDL, very-low-density lipoproteins.

Table 3. Baseline characteristics of patients with RA according to cIMT.

Variable	RA with IMT >p90 n=10	RA with IMT ≤p90 n=30	p Value
Age, years, median (IQR)	55.3 (48.6-68.3)	55.7 (53.3-61.6)	0.900
Female sex; n (%)	6 (60.0)	29 (96.7)	0.002
Smoking			0.818
Never, n (%)	4 (40.0)	11 (36.7)
Exsmoker, n (%)	4 (40.0)	15 (50.0)
Active smoker, n (%)	2 (20.0)	4 (13.3)
Comorbidities
Arterial hypertension, n (%)	3 (30.0)	7 (23.3)	0.673
Diabetes mellitus, n (%)	1 (10.0)	1 (3.3)	0.442
Cardiovascular disease, n (%)	0 (0.0)	3 (10.0)	0.298
Anthropometric characteristics
BMI (kg/m2), median (IQR)	27.1 (24.4-32.2)	26.6 (24.6-29.5)	0.700
Obesity, n (%)
Waist circumference, (cm), median (IQR)	106.5 (87-110.7)	89 (83-103)	0.212
Hip circumference (cm), median (IQR)	106 (103.2-109.7)	106.5 (102.2-112.2)	0.941
Waist-hip index, median (IQR)	0.92 (0.83-1)	0.85 (0.81-0.91)	0.048
MET-minute, median (IQR)	247.5 (70.0-618.7)	594.0 (84.0-1064.0)	0.164
Total MEDAS, median (IQR)	10. (8.7-11.0)	10.0 (8.0-11.0)	0.824
Framingham %, median (IQR)	4.6 (1.5-13.8)	1.2 (0.6-3.8)	0.039
Clinical-laboratory characteristics
Time since diagnosis of RA, months, median (IQR)	140 (93-214.4)	113 (80-166.2)	0.138
Diagnostic delay, months, median (IQR)	9.9 (5.5-18.5)	6.9 (5.3-12.0)	0.414
Erosions, n (%)	4 (40.0)	12 (40.0)	0.473
RF >10, n (%)	7 (70.0)	21 (70.0)	1.000
ACPA >20, n (%)	8 (80.0)	22 (73.3)	0.473
High ACPA (>340), n (%)	6 (60.0)	10 (33.3)	0.036
High-sensitivity CRP (mg/dl), median (IQR)	4.4 (3.2-8.7)	3.8 (2.5-7.5)	0.221
ESR (mm/h), median (IQR)	12.0 (7.7-37.2)	15.0 (9.0-26.0)	0.839
DAS28 at protocol, median (IQR)	3.3 (2.5-3.9)	2.9 (2.5-4.2)	0.644
HAQ, median (IQR)	1.3 (0.7-1.7)	0.8 (0.2-1.6)	0.544
Synthetic DMARDs, n (%)	9 (90.0)	22 (73.0)	0.174
Methotrexate, n (%)	5 (50.0)	18 (60.0)	0.580
Biologic DMARDs, n (%)	4 (40.0)	18 (60.0)	0.271
Corticosteroids, n (%)	6 (60.0)	7 (23.3)	0.032

Abbreviations: cIMT, carotid intima media thickness.

Table 4. Lipid profile and carotid ultrasound in RA patients according to cIMT.

Variable	RA with IMT >p90 n=10		RA with IMT ≤p90 n=30		RA with IMT >p90 vs RA with IMT ≤p90 p
	Fasting	Postprandial	Fasting	Postprandial	Fasting	Postprandial
Fasting lipid profile
Total cholesterol (mg/dl), median (IQR)	226.0 (189.5-243.2)	204.1 (184-237.2)	210.2 (187.2-225.7)	200.0 (179.2-222.2)	0.471	0.573
LDL cholesterol (mg/dl), median (IQR)	138.2 (118.7-152.0)	110.0 (107.2-130.5)	123.5 (105.5-139.0)	110.0 (95.5-129.7)	0.266	0.647
HDL cholesterol (mg/dl), median (IQR)	61.0 (51.2-69.7)	57.5 (49.0-65.7)	66.5 (56.5-77.5)	63.5 (53.7-73.7)	0.045	0.042
Triglycerides (mg/dl), median (IQR)	112.0 (82.7-17.6)	195.0 (127.5-285.2)	77.5 (863.5-107.2)	116.0 (83.5-176.2)	0.014	0.033
Chylomicrons (triglycerides), median (IQR)	33.8 (14.2-57.0) *	79.1 (25.6-167.1) *	14.1 (9.3-23.2)	32.7 (21.8-54.7)	0.066	0.045
Chylomicrons (cholesterol), median (IQR)	12.3 (8.6-16.1)	15.6 (5.7-25.0)	8.7 (6.0-11.6)	15.3 (7.3-24.5)	0.089	0.770
VLDL (triglycerides), median (IQR)	24.2 (21.9-39.5)	42.6 (17.3-60.0)	14.4 (9.8-26.4)	23.1 (13.1-39.7)	0.022	0.036
VLDL (cholesterol), median (IQR)	5.3 (3.9-9.4)	8.2 (3.7-12.7)	2.8 (1.9-6.0)	3.9 (2.7-8.9)	0.028	0.046
ApoB48, median (IQR)	8.5 (5.9-13.0) *	24.3 (15.1-27.1) *	7.7 (5.5- 10.3)	13.5 (10.5-18.2)	0.186	0.017
ApoB total, median (IQR)	102.7 (94.4-115.1)	98.0 (87-110.8)	94 (82.6-104.3)	91.7 (76.1-102.6)	0.141	0.100
Increased postprandial blood lipids
Triglycerides (mg/dl), median (IQR)		73.2 (24.0-134.5)		39.9 (17.2-68.0)		0.122
Chylomicrons (triglycerides), median (IQR)		47.4 (14.6-124.1)		21.5 (10.2-37.7)		0.045
VLDL (triglycerides), median (IQR)		12.9 (6.4-20.7)		8.0 (3.0-16.2)		0.424
ApoB48, median (IQR)		12.3 (10.8-14.3)		6.7 (3.4-8.6)		0.002

* p <0.005 fasting vs postprandial value. Abbreviations: cIMT, carotid intima media thickness; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglycerides; VLDL, very-low-density lipoprotein

 

4. Why secondary Sjögren syndrome was not an exclusion criteria?

R: We did not exclude secondary Sjögren syndrome because it is considered by most authors as part of the same spectrum of rheumatoid arthritis in most patients.

Rferences:

Anaya JM, Rojas-Villarraga A, Mantilla RD, Arcos-Burgos M, Sarmiento-Monroy JC. Polyautoimmunity in Sjögren syndrome. Rheum Dis Clin North Am 2016;42:457–72.

Shiboski SC, Shiboski CH, Criswell L et al. American College of Rheumatology classification criteria for Sjogren’s syndrome: A data-driven, expert consensus approach in the Sjögren’s International Collaborative Clinical Alliance cohort. Arthritis Care Res (Hoboken) 2012; 64; 475–87.

Author Response File: Author Response.pdf

Reviewer 3 Report

Interesting study, requires much effort yet needs minor grammar/spelling check and some comments:

a. suggested edits:

line 45: multiple morbidities

line 47: to both

line 71: plaque burden/ presence

line 77: remove 1st (and), use coma instead

b. comments:

line 91, if possible to be more specific and give clarification of disease symptoms

line 95, sample taken from all patients or all study population ( patients and controls), please  double check when to use patients, controls or study population.

line 121, how many fasting hours were required before checking blood lipid levels? 

line 194, please make sure that median and P value match those in the table, unless it's a different method of calculation.  since median subtraction of apob48 from table 2 doesn't give those results.

I have some conservation regarding the selection and randomaization of controls. Also about external validity. 

 

Author Response

Revisor 3

Comments and Suggestions for Authors

Interesting study, requires much effort yet needs minor grammar/spelling check and some comments:

1. suggested edits:

R: We appreciate your comments and following the recommendation offered by the Reviewer we have changed this point.

line 45: multiple morbidities

R: The authors changed in line 45 “It is associated with premature death and multiple morbidities”

line 47: to both

R: The authors changed in line 47 “Accelerated atherosclerosis in patients with RA is due to both the presence of traditional cardiovascular risk factors and to non-traditional cardiovascular risk factors, including systemic inflammation and dyslipidemia”

line 71: plaque burden/ presence

R: The authors changed in line 71: “The objective of the present study was to analyze the association between postprandial lipid values (i.e., ApoB48 levels after a mixed breakfast) and subclinical atherosclerosis measured as cIMT and plaque presence in patients with RA.”

line 77: remove 1st (and), use coma instead

R: The authors changed in line 77: “The study was carried out at IBIMA, in the Rheumatology Department of Hospital Regional Universitario de Málaga (HRUM), in the Lipid and Atherosclerosis Laboratory of Universidad de Málaga (UMA) and the Center for Medical and Health Research (CIMES), Spain”

1. comments:

line 91, if possible, to be more specific and give clarification of disease symptoms

R: We added in line 91 “The controls were volunteers aged more than 16 years with no inflammatory or autoimmune diseases or symptoms that would lead us to suspect these diseases (i.e. joint inflammation, inflammatory pain, morning stiffness, etc.). The exclusion criteria were the same as for the patients. The controls were selected at random from a health center in the catchment area of the hospital. Controls and patients were matched by age and sex."

line 95, sample taken from all patients or all study population (patients and controls), please  double check when to use patients, controls or study population.

R: The authors changed in line 95: “The study population then had a mixed breakfast in the hospital cafeteria (milk, cured ham, cheese, olive oil, and bread).”

line 121, how many fasting hours were required before checking blood lipid levels? 

R: The study population fasted 8 hours

We added in line 125-127: “ApoB48 and total ApoB levels were measured in plasma after 8 hours fasting and 4 hours after the meal using a commercially available ELISA approach (Shibayagi Co Ltd, Ishihara, Japan)”

line 194, please make sure that median and P value match those in the table, unless it's a different method of calculation.  since median subtraction of apob48 from table 2 doesn't give those results.

R: The authors apologize for this mistake in text. The data in the table is correct.

We have changed the text in line 207-208: “The increase in ApoB48 was significantly greater in patients than in controls (median [IQR], 14.4 [10.8-23.2] vs 12.1 [10.9-16.2]; p=0.042).”

I have some conservation regarding the selection and randomization of controls. Also, about external validity. 

R: Our patients were consecutively included from our RA consultations whilst controls were chosen by simple randomization of a listing and then they were invited to participate. Our sample of patients is the usual one in routine clinical practice, patients undergoing the same treatments and comorbidities as in clinical practice, so we think that these results can be generalized to other patients with RA.

Round 2

Reviewer 2 Report

No further comments