More than 70 million individuals within the United States alone currently live with cardiovascular disease 1, while nearly 21 million people have diabetes and 54 million are diagnosed with pre-diabetes 2. Oxidative stress is suggested as one unifying link between cellular dysfunction and the onset and progression of both cardiovascular 3 and metabolic 4 disorders. Although a basal level of radical production is important for proper physiological function 5, overexpression of radical species, either via increased production or decreased antioxidant defense, can result in oxidative damage to lipids, proteins, and nucleic acids, all of which may contribute to the pathophysiology of disease 6. In particular, oxidative damage to the vascular endothelium is well described 7 and is associated with a poor cardiovascular prognosis 8.

Meals high in fat and carbohydrate that lead to hypertriglyceridemia or hyperglycemia have been shown to promote an exacerbation in radical production 9 and have been associated with increased risk for atherosclerosis and related disorders 10. Thus, postprandial oxidative stress (and associated measures of triglycerides and blood glucose) appears to provide more important information related to cardiovascular and metabolic disease development and progression 311.

Individuals with impaired glucose metabolism are most susceptible to postprandial oxidative stress, as they typically experience prolonged periods of hyperglycemia 121314 and hypertriglyceridemia 1516 post feeding. Elevations in blood glucose and triglycerides are directly associated with superoxide anion production 17, a potent and harmful radical species 18.

Antioxidant intake has been used in several studies in an attempt to attenuate the rise in postprandial oxidative stress. This body of work has included the study of foods such as olive oil 19, almonds 20 and red wine 21, as well as nutritional supplements containing vitamins and minerals 222324. One antioxidant nutrient with a significant body of literature in support of its antioxidant effects is carnitine 2526 which is associated with a reduction in xanthine oxidase activity 27 and known to possess free-radical scavenging activity 28.

A recent report by Volek and colleagues 29 demonstrated the potential benefits of L-carnitine, L-tartrate supplementation on endothelial cell function, assessed via flow mediated dilation (FMD). The authors suggest a potential role of nitric oxide in promoting these findings, as nitric oxide is a signaling molecule known to facilitate vasodilatation by acting on vascular smooth muscle cells 30. This appears partly supported by preliminary studies involving carnitine supplementation and measurement of nitric oxide in a fasted state 313233. However, to our knowledge, no study has investigated the potential benefits of dietary carnitine either alone or in conjunction with L-arginine (the precursor to nitric oxide biosynthesis 34) on circulating nitric oxide during the postprandial state, and only that of Volek et al. 29 has included a measure of postprandial oxidative stress (malondialdehyde). Because nitric oxide typically decreases in response to an increase in postprandial oxidative stress 2235, likely mediated by the increase in superoxide production and associated endothelial dysfunction 36, maintenance of nitric oxide via carnitine ingestion could be viewed as favorable in regards to vascular function. This may be of particular importance for those with metabolic dysfunction.

Therefore, the purpose of this study was to determine the effects of supplementation with a novel L-arginine containing form of acetyl L-carnitine supplementation (ArginoCarn^®) on postprandial oxidative stress and associated metabolic biomarkers in a sample of pre-diabetic men and women. We hypothesized that 1) oxidative stress biomarkers would be increased, and nitric oxide decreased, following ingestion of a high fat, high carbohydrate test meal, and 2) subjects assigned to the arginine/carnitine supplement for eight weeks would experience attenuation in postprandial oxidative stress and nitric oxide changes.

Although 40 subjects were initially enrolled in the study, only 29 subjects were included in the data analysis due to failure to complete the entire eight weeks of the intervention or a request to be dropped from the study. It should be noted that no subject was dropped due to an adverse outcome associated with the ALCA or placebo supplementation. Rather, loss of interest and personal issues unrelated to the study protocol was the chief reason for subject attrition. Of the 29 subjects remaining, compliance to treatment was 93% for ALCA and 91% for placebo, and not different between conditions (p > 0.05).

No differences were noted between conditions or from pre to post intervention in anthropometric variables, resting heart rate or blood pressure, or fasting metabolic blood parameters (Table 1; p > 0.05; Figures 1, 2, 3). The only exception was higher fasting nitrate/nitrite noted for ALCA compared to placebo post intervention (30.1 ± 2.8 vs. 23.6 ± 2.1 μmol·L^-1; p = 0.01). This was influenced by the response of a portion of those subjects receiving ALCA, as only eight of the fourteen subjects showed an increase (greater than 5%) in fasting nitrate/nitrite values from pre to post intervention (Figure 4). No differences were noted regarding intake of total kilocalories, total grams of protein, carbohydrate, fat, vitamin C intake, vitamin E intake, or vitamin A intake (Table 2; p > 0.05). No statistically significant differences (p > 0.05) were noted between conditions or pre-post intervention in the AUC for malondialdehyde (Figure 1A), xanthine oxidase activity (Figure 1B), hydrogen peroxide (Figure 1C), triglycerides (Figure 2A), glucose (Figure 2B), TEAC (Figure 3A) or nitrate/nitrite (Figure 3B). Individual subject data for percent change from pre to post intervention for fasting nitrate/nitrite values are presented in Figure 4. All effect size calculations were trivial (r < 0.1) except for nitrate/nitrite from pre to post intervention for ALCA, which was considered moderate (r = 0.35). AUC data for all variables are shown in Table 3.

Findings from this investigation indicate that eight weeks of supplementation with ALCA results in an increase in resting nitrate/nitrite in pre-diabetics, but does not promote significant changes in other metabolic or oxidative stress variables measured at rest or post meal. The increase in fasting nitrate/nitrite was influenced primarily by eight of the fourteen subjects receiving ALCA (Figure 4), highlighting the variability in response to dietary supplementation with L-carnitine in regards to blood nitrate/nitrite, a finding we have previously noted using glycine propionyl-L-carnitine 33. In reviewing subject characteristics in an attempt to determine reasons for the discrepancies in subject response, we noted no apparent differences between "responders" and "non-responders" that would lead us to suggest an association, with the possible exception of slightly lower starting values for some, but certainly not all responders. In this case, it is possible that individuals with lower nitrate/nitrite values may respond to a greater extent to ALCA treatment. Future work is needed to more fully elucidate the reasons for such discrepancies in individual response to ALCA treatment in regards to fasting nitrate/nitrite levels. It is possible that a portion of the increase in blood nitrate/nitrite could have been due to an increase in physical activity of certain subjects assigned to ALCA, as an increase in resting nitrate/nitrite has been reported previously for individuals involved in exercise training 545556.

Based on the collective data we must reject our hypothesis that ALCA would lead to favorable effects on postprandial oxidative stress and associated biomarkers. However, our findings of a moderate effect size for increase in the AUC for nitrate/nitrite from pre to post intervention with ALCA may have clinical relevance, despite the lack of statistical significance. Of course, this hypothesis needs further investigation, and needs replication in a larger sample of human subjects; in particular in regards to identifying those individuals who are responders to treatment and those who are not.

Unlike the recent work of Volek and coworkers 29 that included only one measure of oxidative stress (malondialdehyde), we included a full panel of commonly assessed oxidative stress biomarkers within the present design. Our failure to note any significant effect of ALCA in regards to this panel provides convincing evidence that no treatment effect related to postprandial oxidative stress is noted with supplementation, at least at the dosage (3 g·day^-1) and for the duration (8 weeks) provided. This, despite the fact that our chosen treatment included L-arginine, a known precursor to nitric oxide biosynthesis 34, as one component of the supplement. Whereas, the study by Volek and colleagues used L-carnitine, L-tartrate as the supplement of choice 29.

Our data related to nitrate/nitrite provide partial support for the findings of improved FMD with carnitine intake 29, as nitric oxide is known to facilitate vasodilatation by acting on vascular smooth muscle cells 30. Such an effect may be associated with favorable clinical outcomes, as endothelial dysfunction is linked to cardiovascular morbidity and mortality 8. However, it is unknown what, if any, clinical relevance both our findings and those of Volek et al. 29 may have. This is particularly true for the extremely small, but statistically significant findings of Volek et al. 29, who noted an increase in postprandial (1.5 hour) FMD from 6.6% to 7.7% with carnitine treatment as compared to a decrease in FMD from 6.6% to 5.8% with placebo. While these findings were noted as statistically significant, their clinical relevance must be questioned, especially considering the somewhat subjective nature of this brachial artery imaging assessment. Moreover, no other differences were noted between carnitine and placebo at the 3.0 and 4.5 hour postprandial measurement times. In fact, FMD was actually higher for placebo at these times compared to carnitine. Clearly, replication of these findings is needed before firm conclusions can be made regarding the impact of supplemental carnitine intake on vascular health and function. This is particularly true considering that no other measures in this study by Volek and colleagues 29 were noted to be significantly effected by carnitine (e.g., triglycerides, malondialdehyde, insulin, interleukin-6, tumor necrosis factor-alpha).

Carnitine may promote increased or maintained nitric oxide in two distinct manners. First, eNOS gene expression has been demonstrated to be increased within cultured human endothelial cells following carnitine incubation 25. We have recently reported in two studies using human subjects that oral supplementation with carnitine (glycine propionyl L-carnitine) increased nitrate/nitrate at rest 32 and in response to static exercise 33. Lofreddo and coworkers 31 have also demonstrated that intravenous injection of propionyl L-carnitine increases nitrate/nitrate in patients with peripheral arterial disease. Second, the antioxidant effects of carnitine can provide protection against nitric oxide destruction via superoxide production 57. L-carnitine has been shown to have effective hydrogen peroxide and superoxide anion scavenging abilities 58, which may minimize the interaction between nitric oxide and superoxide, ultimately leading to decreased formation of peroxynitrite 57 and maintenance of nitric oxide. Hence, carnitine may lead to both the production of nitric oxide, as well as the maintenance of existing nitric oxide.

Our hypothesis for attenuation in oxidative stress biomarkers with ALCA treatment was based on a sound body of literature demonstrating antioxidant benefits of carnitine in both humans 325960 and animals 276162. Our failure to note significant antioxidant effects related to our chosen biomarkers could be explained in at least two manners.

First, it is possible that the dosage of ALCA used in the present study, in conjunction with the relatively large nutrient load, was not adequate to provide protection against the massive oxidative insult incurred by the test meal. Most previous studies have simply measured oxidative stress biomarkers in a rested, fasted state following carnitine administration. Our use of the test meal may have overwhelmed the antioxidant ability of the ALCA, leading to our null results. This is particularly true considering that our test meal, although not of uncommon size and composition for meals consumed by many overweight/obese individuals, was relatively large compared to some previous studies 636465. This may have lead to increased radical production, possibly minimizing any positive effects of the ALCA treatment.

Second, our sample consisted of pre-diabetics, thus deficiencies in postprandial glucose and lipid metabolism likely occurred in response to the test meal. Surprisingly, however, we noted only a minimal blood glucose response to feeding. This is likely due to both the timing of our measurements (i.e., first measurement occurring one hour post feeding), as well as the inclusion of a high amount of dietary fat to the test meal. Almost identical findings have been reported recently by Blendea and colleagues 66. In addition to being pre-diabetic, our subjects were overweight or obese. We have recently noted that obese individuals experience an exaggerated lipemic and oxidative stress response to high fat, high carbohydrate feeding, as compared to normal weight subjects 45. This increase in blood triglyceride following feeding is directly linked to superoxide production 6768, likely mediated by hypertriglyceridemic-induced neutrophilia 69. Circulating triglycerides may also result in accelerated superoxide production from the abundance of substrate processing and subsequent production of reducing molecules (e.g., NADH, FADH₂) generated with our large meal 70. Obese individuals also have lower plasma antioxidants and higher tissue lipid levels which can lead to an increased susceptibility to oxidative stress 71. Hence, our sample of pre-diabetics may have experienced too great an increase in radical production following the meal, further hampering the ability of the ALCA to provide measurable antioxidant effects. Future work may consider the use of 1) a higher dosage of ALCA, 2) smaller test meals, and 3) the use of metabolically normal test subjects in order to further determine the potential antioxidant benefits of ALCA in relation to postprandial oxidative stress and associated biomarkers.

Eight weeks of supplementation with ArginoCarn^® results in an increase in resting nitrate/nitrite in pre-diabetic subjects, which may allow for higher overall circulating nitrate/nitrite following intake of a high kilocalorie, high fat and carbohydrate meal. This ingredient also results in a slight improvement in glucose metabolism, as evidenced by minor decreases in blood glucose, insulin, and HbA1c (which may have been more pronounced with longer treatment, as HbA1c has a half-life equal to approximately 12 weeks 72). However, ALCA does not result in significant changes in other metabolic or oxidative stress variables measured at rest or after feeding. It is possible that a greater dosage and/or duration of treatment of ALCA is needed for attenuation of postprandial oxidative stress. Moreover, such factors as the size and composition of the test meal, in addition to abnormal glucose metabolism and excess body weight/fat of our test subjects, may have contributed to the null findings. That is, any potential antioxidant effect of ALCA may have been overwhelmed by the oxidant burden of the test meal, coupled with potentially exacerbated radical generation and decreased antioxidant defense of our test subjects. The increase in nitrate/nitrite may indicate positive effects related to vascular function. Of course, additional study is necessary to confirm this hypothesis.

Funding for this work was partially provided by Sigma-tau HealthScience. The authors nor the University of Memphis directly endorse the dietary supplement used in this investigation. RJB has been the recipient of research support from the project sponsor since 2005, and is periodically involved in scientific writing for the sponsor.

RJB was responsible for the study design, biochemical work, statistical analyses, and manuscript preparation; KHFW and PST were responsible for data collection, blood collection and processing, and manuscript preparation. All authors read and approved of the final manuscript.