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 . 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 [54–56].
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  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 , as one component of the supplement. Whereas, the study by Volek and colleagues used L-carnitine, L-tartrate as the supplement of choice .
Our data related to nitrate/nitrite provide partial support for the findings of improved FMD with carnitine intake , as nitric oxide is known to facilitate vasodilatation by acting on vascular smooth muscle cells . Such an effect may be associated with favorable clinical outcomes, as endothelial dysfunction is linked to cardiovascular morbidity and mortality . However, it is unknown what, if any, clinical relevance both our findings and those of Volek et al.  may have. This is particularly true for the extremely small, but statistically significant findings of Volek et al. , 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  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 . We have recently reported in two studies using human subjects that oral supplementation with carnitine (glycine propionyl L-carnitine) increased nitrate/nitrate at rest  and in response to static exercise . Lofreddo and coworkers  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 . L-carnitine has been shown to have effective hydrogen peroxide and superoxide anion scavenging abilities , which may minimize the interaction between nitric oxide and superoxide, ultimately leading to decreased formation of peroxynitrite  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 [32, 59, 60] and animals [27, 61, 62]. 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 [63–65]. 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 . 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 . This increase in blood triglyceride following feeding is directly linked to superoxide production [67, 68], likely mediated by hypertriglyceridemic-induced neutrophilia . 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 . Obese individuals also have lower plasma antioxidants and higher tissue lipid levels which can lead to an increased susceptibility to oxidative stress . 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.