To the best of our knowledge, this study is the first meta-analysis of RCT to examine the effect of whey supplementation on circulating CRP level. Our primary result showed that supplemental whey protein and its derivates were insufficient to change the circulating CRP level. However, the supplementation produced a significant CRP reduction among participants with high supplemental doses or increased baseline CRP.
This meta-analysis followed the PRISMA guidelines and had a relatively high Jadad score. However, it was primarily limited by considerable heterogeneity across studies, which complicated the interpretation of our findings. This is not surprising, given the variation in study characteristics. In this meta-analysis, intervention included whey protein, hydrolyzed whey protein and whey peptides. Each component from them may function differently in terms of inflammatory response. The participants also included overweight/obese adults, as well as patients with hypertension, COPD or metabolic syndrome. Healthy status may differently influence the effect of whey protein on inflammatory response. In addition, genetic background or a gene-diet interaction may have been sources of heterogeneity across studies. Although we cannot further investigate the effect based on these characteristics because of the limited number of trials, the observed heterogeneity could be attributed to the two following trials, because heterogeneity disappeared after each of these trials was excluded in the sensitivity analyses. In the trial of Pal, the disparate results were likely due to the high daily dose of whey. This trial is the only one with a statistically significant CRP reduction [21
]. In the trial of Gouni-Berthold, the baseline CRP level is only 0.4 mg/L. This value was the lowest among the trials. This low value may be partly attributable to the two months’ dietary and lifestyle recommendations before the intervention, because this healthy recommendation may decrease the CRP level. The CRP-reducing effect became statistically significant after this trial was excluded [16
Subgroup analysis results indicated the influence of daily whey dose on the change in CRP. We found that CRP reduction was more pronounced when whey supplementation was ≥20 g/day, suggesting that whey quantity is an important factor affecting CRP responses. Weinheimer reviewed clinical trials and considered that ≥35 g of whey protein per day is possibly necessary to enhance the effect on metabolic health responses [19
]. In theory, the consumption of any dairy products by adults results in an almost complete breakdown of protein to peptides and to the amino acid level. Indeed, the susceptibility of whey protein and its derivates to intestinal enzymes is a major problem that complicates the therapeutic use of such proteins [17
]. Thus, a high dose of whey protein may be required to elicit a measurable biological effect in humans.
Subgroup analysis results also indicated a significantly larger reduction of CRP in subjects with increased CRP (≥3 mg/L) at baseline. This finding was also supported by meta-regression analysis that baseline CRP was a major source of heterogeneity among trials. This result is important, because more than 3 mg/L of CRP results in a high risk of future CVD events [5
]. A decrease in CRP is also helpful to alleviate T2DM, because the CRP level is positively associated with T2DM incidence [24
]. Obesity, which is an established risk factor for CVD and T2DM, has been associated with elevated levels of CRP [25
]. Thus, our finding is useful, because intervention could be effective in individuals who most needed this treatment.
In the subgroup analysis, a significant decrease of CRP was found in CRP and not in hsCRP. However, we should mention that hsCRP is the same kind of protein as CRP. CRP is called hsCRP when a highly-sensitive assay is used. In fact, hsCRP assays were commonly available after 2000 [26
]. Some researchers still used the term CRP in the study, even if a highly-sensitive assay were used. Thus, the difference between CRP and hsCRP should be interpreted with caution because of a lack of unified nomenclature used in these studies.
Despite the insufficient evidence provided in the present study, whey protein possibly exhibited a potential CRP-lowering effect. CRP is principally induced by interleukin (IL)-6 and IL-8 via a mechanism involving the activation of the nuclear factor kappa B pathway, which is a key regulator of pro-inflammatory mediator synthesis [27
]. Intense exercise generally induces increased levels of pro-inflammatory mediators [30
]. The consumption of cake consisting of whey protein significantly reduces the exercise-increased CRP and IL-6 by 46% and 50%, respectively [31
]. Cystic fibrosis is characterized by chronic pulmonary inflammation. Pressurized whey supplementation significantly decreases the increased CRP and IL-8 levels in these patients [32
]. These two trials were excluded in the meta-analysis because of the acute intervention in the former and the lack of a control group in the latter. The effects of whey protein on inflammation are not limited to CRP. Several RCTs used other inflammatory markers, such as IL-6, IL-8 and TNF-α. For example, Sugawara found that circulating levels of IL-6, IL-8 and TNF-α in the patients with COPD significantly decreased after whey intervention compared with those in the control group [18
]. However, no significant change in IL-6 and TNF-α was observed in overweight individuals between the whey group and control group [21
Inflammation status and oxidative stress phenomena are narrowly interacting in disorders, such as obesity, CVD and T2DM [33
]. Whey protein is rich in cysteine, which can increase the synthesis of glutathione, a crucial intracellular antioxidant [34
]. In one trial included in this meta-analysis, the change in glutathione and CRP levels after pressurized whey protein was consumed by COPD patients was not observed [18
]. However, another trial found that the pressurized whey protein supplementation of 45 g/day in healthy adults increased lymphocyte glutathione by 24% [35
]. Likewise, our animal study observed that the plasma glutathione level was significantly higher in rats fed with HFD adding 15% whey protein than HFD-only fed rats [2
]. A further in vitro
study demonstrated that 6.24 mg/mL of whey protein increased the glutathione level by 138% in C2C12 muscle cells that induced oxidative stress by tert
-butyl hydroperoxide [36
]. Whey protein contains a high level of other essential amino acids, such as leucine. An animal study from our laboratory also confirmed that 1.6% leucine supplementation with 15% whey protein significantly enhanced the antioxidant capacity in non-obese insulin resistant rats [2
]. In a clinical trial with overweight or obese subjects, administration of nutraceutical containing 2.25 g of leucine per day for four weeks significantly reduced oxidative and inflammatory biomarkers (TNF-α, CRP) and increased the anti-inflammatory marker, adiponectin [37
]. Furthermore, milk-derived bioactive peptides exert beneficial effects on the prevention and treatment of chronic metabolic diseases via multiple mechanisms, such as regulation of insulin resistance and blood pressure, affecting the oxidative stress levels and alteration of the lipid profiles [3
Finally, evidence from recent years also suggests that milk casein per se
can actively affect the inflammatory process with inconsistent findings. Aihara found that oral administration of milk casein-derived tripeptide Val-Pro-Pro for 10 weeks exerts an anti-inflammatory effect on the adipose tissue of HFD-fed mice [39
]. Hirota reported that casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro supplementation in subjects with mild hypertension significantly reduced circulating TNF-α levels, although no alteration in circulating CRP level was found [40
]. Similarly, no alterations in circulating CRP levels were observed in overweight adolescents supplemented with casein protein for a total of 12 week [41
]. Therefore, more trials are required to further investigate the relationship between casein intake and inflammatory biomarkers.