Reply to the Letter to the Editor from J. Matthews and C. Sale

Dear Editor We thank Joseph Matthews and Craig Sale for their interest in our research and their comments, and we are thankful for the opportunity to respond to the issues raised in their letter. The first point raised refers to the combining of HCD precursors (β-alanine and histidine) with HCDs (since 17 out of the 30 studies did not supplement with HCDs). It was also pointed out that while supplementation with β-alanine may increase skeletal muscle HCD content, supplementation with histidine does not alter HCD content in humans and that both precursors have biological actions independent from their role in HCD metabolism. There was concern that one study which used only histidine supplementation had a large moderating effect on the weighted mean difference for changes in waist circumference. We agree with Matthews and Sale that β-alanine increases skeletal muscle HCD concentration as has been demonstrated in previous studies. We also know that there is evidence to suggest β-alanine increases expression of glucose transporters in cell culture. One study exploring the effect of histidine on muscle carnosine suggested that even if histidine supplementation failed to directly increase muscle carnosine concentrations in humans, chronic β-alanine supplementation in humans may gradually deplete muscle histidine via a failure of dietary intake to match the increasing use of histidine (semiessential, proteinogenic amino acid) for carnosine synthesis. If that is true, histidine availability could become rate limiting for carnosine synthesis under conditions of chronic β-alanine use, and increase in muscle carnosine would be more efficient when chronic β-alanine supplementation is combined with histidine. Evidence also shows that histidine has anti-oxidative and anti-inflammatory effects, suggesting a possible class effect. In addition, some effects of carnosine are ascribed to the effects of histidine. For these reasons, histidine was included in the meta-analysis. Secondly, Matthews and Sale state that of the 13 studies which supplemented with HCDs, five included carnosine in combination with other ingredients, and in three studies, carnosine was not the primary ingredient, supplemented alongside either cinnamon or α-lipoicacid, both of which have purported therapeutic effects. There was also concern that the amount of carnosine ingested in each study (165, 200, and ~585mg.d-1) is insufficient to raise plasma or skeletal muscle HCD concentrations making it difficult to ascribe any of the beneficial outcomes to HCDs alone, especially given that they had a large moderating effect on the weighted mean difference for changes in fasting glucose and HbA1c. We acknowledge the heterogeneity among the included studies and their use of co-interventions alongside carnosine or HCDs. We also recognize that previous literature suggests the necessary dose for a biological effect to be 500–3500 mg. The dose of ~585 mg mentioned above in the study by Karkabounas et al. is the dose per tablet taken three times a day giving an approximate dose of ~1700 mg/day. Because we use aggregate meta-analysis rather than individual data, it is not possible to determine from the available data whether there are dose–response effects or whether the effects can be solely ascribed to carnosine, other HCDs, or other additives, and we have listed this as a limitation of the review, highlighting the need for further studies to confirm our findings (page 11, section 4.3.). Given the limited number of HCD studies in humans, the inclusion of these studies, despite their heterogeneity, was intended to provide an overview of the current literature and the collective reported effects, including an understanding of the limitations within this literature. A third comment made by Matthews and Sale is that if the data for waist circumference, fasting glucose, and HbA1c were re-analysed, removing studies that supplemented histidine in isolation or supplemented carnosine alongside other ingredients, the effect of the intervention is no longer significant. They also raised concern that the quality for the three main outcomes (determined by the GRADE approach) was ‘low’ or ‘very low’ therefore having ‘limited’ or ‘very little’ confidence in the effect estimate undermining the statements made in the manuscript title and conclusions. It is not surprising that re-analysis of a different set of studies, particularly that of a smaller number of studies, would alter the results. The purpose of including all studies in our main analysis, as mentioned previously, was to provide an inclusive overview of the available literature and the reported impact on the outcomes of interest. Again, as noted in the manuscript discussion, we highlight the heterogeneity of the included studies as a limitation to our findings (page 11, section 4.3), and we emphasize the need for further studies to confirm these results and strengthen the evidence base. This is noted throughout the discussion (end of paragraphs 1 and 3 in section 4.2 and in section 4.3) as well as in the conclusions (section 5). Indeed, as mentioned by Matthews and Sale, we have also expressed this in our GRADE evaluation, where we describe the evidence as being of ‘low’ and ‘very low’ quality, reiterating that the evidence is of poor quality and that the results should be interpreted in light of the above limitations. The final point raised is that the study outcomes from this review were focused on anthropometric and glycaemic outcomes which was a deviation from our previously published protocol which states study outcomes as ‘All chronic disease risk factors and outcomes including but not limited to metabolic/glycaemic, cardiovascular and cognitive/mental health . ’. The concern raised by Matthews and Sale is that selective outcome reporting is a methodological concern and can introduce bias into the analyses based on results that return Received: 14 April 2020 Accepted: 16 April 2020