Previous work had suggested that a shift in the diet to lower Lys:Arg ratios had a favorable effect on cardiovascular risk factors in animal models and humans. By design, the intent of this study was to evaluate diets with different amino acid profiles, rather than focusing on a single amino acid or type of protein. This end was achieved by using commercially available food products and without amino acid supplements. Taking this approach the Lys:Arg ratio was shifted from 0.7 to 1.4. Nuts and legumes displaced foods containing animal protein from the diet to decrease the Lys:Arg ratio. By substituting nuts and legumes for animal protein the Arg content of the low Lys:Arg diet was 1.5-times higher than in the high Lys:Arg ratio diet. For moderately hypercholesterolemic participants, the difference in Lys:Arg ratio resulted in a null or small effect on cardiovascular disease risk factors. When differences were observed they were, for most part, in the non-fasting state.
From observational data, daily Arg intake has been estimated to range between 4.3 and 13.8 g/d, dictated by different regional dietary habits [61
]. The Arg intake of our participants ranged from 8–12 g/day and 5–8 g/day, when consuming the low Lys:Arg and high Lys:Arg ratio diets, respectively, depending on energy requirements needed to maintain a stable body weight. These values are well within the range from observational studies.
The difference in Lys:Arg ratio between the two diets was large in terms of the ratio and what could be reasonably achieved using habitually consumed foods alone (0.7 vs. 1.4), yet less than that used in animal studies. Differences in Lys:Arg ratios among studies involving animal models range from 0.3 to 2.2. In contrast to the current work, these animal studies reported a significant effect of Lys:Arg ratio on lipoprotein concentrations. For example, Sprague-Dawley rats fed a high Lys:Arg ratio (1.58 ratio) diet had the highest, whereas those fed a low Lys:Arg ratio (0.36 ratio) had the lowest total and HDL-cholesterol concentrations [31
]. Similar results were observed in male albino rats (low Lys:Arg ratio, 0.67; high Lys:Arg ratio, 2.0) [32
] and rabbits (high Lys:Arg ratio, 2.2 ratio; low Lys:Arg ratios, 0.9 and 0.3) [33
]. In all these cases, the fatty acid profile of the diets did not differ as a result of manipulating the amino acid profile of the diets. Notably, these studies were conducted using animal species that lack analogies with human cholesterol and lipoprotein metabolism [63
Several studies using rats and rabbits also focused on the effects of other amino acids and suggested that the sulfur-containing amino acids, methionine (Met) and cysteine (Cys), were also hypercholesterolemic, whereas glycine (Gly) was hypocholesterolemic [35
]. Similar to the low Lys:Arg ratio, proteins having a low Met:Gly ratio were purported to elicit a hypocholesterolemic effect [67
]. Of note, in the present study the low Lys:Arg ratio diet had a Met:Gly ratio of 0.32, whereas the high Lys:Arg ratio diet had a Met:Gly ratio of 0.67. These values are comparable to those reported for soy protein and casein (Met:Gly ratio 0.32 and 0.64, respectively).
Postprandial triglyceride and apo AI concentrations were significantly lower at the end of the low Lys:Arg ratio diet phase. However, this difference in triglyceride concentrations was not reflected in higher HDL cholesterol concentrations, as is frequently observed [68
]. The small absolute difference in apo AI concentration is of questionable clinical significance. Furthermore, no significant differences were observed in LCAT or CETP activities at the end of diet phases. There is no obvious explanation for this observation. We cannot rule out the possibility that different sources of carbohydrates and fiber in the two diets may have contributed to the observed postprandial triglyceride concentrations. Interestingly, the effect on triglyceride concentrations was not observed in the fasting state, which suggests an effect on chylomicron-triglyceride, rather than VLDL-triglyceride clearance. It is possible that consumption of the low Lys:Arg ratio diet may have induced greater postprandial insulin secretion relative to the high Lys:Arg ratio. Moreover, the concentration of adiponectin, known to affect triglyceride concentrations and increase insulin sensitivity [69
], was significantly higher, albeit modestly, at the end of the low Lys:Arg ratio diet phase compared to the high Lys:Arg ratio diet phase. It is possible that the higher adiponectin concentrations resulting from the low Lys:Arg ratio diet may have contributed to the difference in insulin sensitivity and enhanced triglyceride clearance despite the lack of differences in fasting insulin concentrations.
The consumption of diets with different Lys:Arg ratios or absolute amounts of Arg had no significant effect on endothelial function assessed by measuring FMD or PAT. Reports suggesting that endothelial function was improved in humans by supplementary L-Arg were at intake levels for the amino acids that far exceeded the amount that could be achieved by manipulating dietary sources of protein as attained for this study. These observations have been made in control participants (21 g L-Arg/d for 4 weeks) [70
], coronary heart disease patients (21 g L-Arg/d for 3 days) [41
], and hypertensive patients (single dose of 6 g L-Arg) [44
]. Notably, studies reporting an effect of supplementary L-Arg on FMD were of much shorter duration than the present intervention, and more likely represent acute effects of L-Arg supplementation. Conversely, more modest doses of supplemental L-Arg failed to have a significant effect on endothelial function in peripheral arterial disease patients (3 g L-Arg/d for 6 months) [71
] and hypercholesterolemic patients (2 bar per day with 3.3 g L-Arg/bar for 1 week) [72
] with the exception of one study involving hypercholesterolemic individuals (2 bar per day with 3.3 g L-Arg/bar for 1 week) in which a significant improvement was reported [73
]. In contrast to the current work, studies in which L-Arg was observed to have an effect on FMD, supplementation resulted in an Arg intake greater than what is consumed through dietary protein alone and the potential effect of dietary lysine was not taken into consideration. Moreover, it has been suggested that the amount of dietary arginine that enters the NO synthesis pathway is relatively low and therefore is unlikely to affect endothelial function [74
In the current study, both fasting and postprandial hsCRP concentrations were significantly lower after the low Lys:Arg ratio diet phase. Consistent with this finding, observational data from the Third National Health Nutrition and Examination Survey (NHANES III) suggested an association between higher consumption of Arg-rich foods and lower serum hsCRP concentrations [75
]. Despite the statistically significant difference observed in the current study, both mean hsCRP concentrations were within the normal range at the end of each diet phase. At this time there is a dearth of data on the effect of dietary amino acids or protein types on inflammatory markers. Additional data are needed prior to adequately interpret these findings.
We cannot rule out the possibility that factors associated with different dietary protein sources or phytochemicals were responsible to the differences in cardiovascular risk factors previously reported. Likewise, we cannot eliminate the possibility that undetected differences in the fatty acid profile of the diets in prior work had a significant effect on the outcome measures. In the current study we attempted to minimize differences between the two diets beyond that of the Lys:Arg ratios. However, due to constraints in the sources of amino acids and the intent to manipulate the Lys:Arg ratio with commonly consumed foods rather than amino acid supplements, we may have impacted study outcomes in unrecognized ways. Despite efforts towards maintaining comparable macronutrient and fiber contents between both diets, the lower Lys:Arg ratio diet had more dietary fat (31.8% energy vs. 25.1% energy) and less dietary fiber (17.8 g/1000 kcal vs. 21.5 g/1000 kcal) than the higher Lys:Arg ratio diet. We cannot rule out the possibility that changes due to these differences may have masked the absolute effect of differences in the Lys:Arg ratio. Lastly, whereas Lys is an essential amino acid, one that humans cannot synthesize adequate amounts to meet requirements, Arg is not. We were unable to determine the potential contribution of endogenous Arg synthesis to the total pool and how this may have affected the Lys:Arg ratio.
In conclusion, the amino acid profile of the diet, characterized by its Lys:Arg ratio, had no effect on fasting cardiovascular risk factors, vascular reactivity, or fractional cholesterol synthesis rate in mildly hypercholesterolemic adults. Overall, no convincing evidence was found to support the positive or negative modulation of cardiovascular disease risk factors based on characterizing dietary proteins by the Lys:Arg ratio.