Quantification and physiological significance of the rightward shift of the V-slope during incremental cardiopulmonary exercise testing

The basic exercise data are summarized in Table 2.

Data are presented as mean ± SD. SBP indicates systolic blood pressure; VO₂, indicates minute oxygen uptake; RR, respiration rate; RER, respiratory exchange rate; VAT, ventilatory anaerobic threshold; VCO₂, minute carbon dioxide production; RtShift, rightward shift of V-slope; IQR, interquartile range.

First, typical examples of a case with very little rightward shift (type A) and a moderate shift (type B) of the V-slope are shown in Fig. 3(a, b). Next, the maintenance of RtShift during a ramp protocol (Fig. 4(a)) is contrasted with its lack during a steady-state protocol (Fig. 4(b)) consisting of a V-slope during three six-min steps. The rightward shift is noted to occur only at the start of the step increase, ultimately reverting back later.

Among the visual method and the two mathematical methods for estimating RtShift, the first method (visual) yielded the highest mean RtShift, followed by the second (quadratic curve fitting) and then the third (simple averaging) method. The mean RtShift values were as follows: first, 50.8 ± 41.6; second, 42.0 ± 34.4; third, 33.9 ± 25.0 mL⋅min⁻¹ VO₂ (each different from the others, p < 0.001). However, the RtShift of each method correlated well with each other (first vs. second, r = 0.955; second vs. third, r = 0.989; first vs. third, r = 0.945). We decided to adopt the third method (simple averaging) to determine RtShift because there was no failure associated with the determination; however, with the second method, there was an 11% failure rate in determining RtShift. The third method also utilized all data points.

As mentioned in the Statistical analysis section, RtShift did not have a Normal distribution (Additional file 3: Figure S3(a)).

VAT (mL⋅min⁻¹) and RtShift (mL⋅min⁻¹ VO₂) were significantly correlated (r = 0.718, p < 0.001; adjusted for weight, r = 0.543, p < 0.001); the greater the VAT, the greater the rightward shift (Fig. 5). VAT was also significantly correlated with RtShift after correction for the resting RtShift (r = 0.402, p < 0.001).

VAT, resting RtShift, and resting end-tidal CO₂ concentration were each significant (p < 0.001, p < 0.001, and p = 0.003; standardized correlation coefficient: 0.5699, 0.3966, and −0.2032, respectively). The univariate correlation between RtShift and resting end-tidal CO₂ concentration was, however, only 0.094 (p = 0.351). The use of a β-blocker was not a significant variable in this analysis (p = 0.612).

Both VAT and RtShift were significantly correlated with ΔVO₂/Δwork rate, a marker of oxygen uptake efficiency (10 ⋅Wmin⁻¹, n = 83; r = 0.531, p < 0.001; adjusted for weight, r = 0.393, p < 0.001, respectively) (Additional file 3: Figure S3(b)).

The result of the hypothesis testing that S1 is parallel to the diagonal R = 1 was as follows: the first test of the mean S1 slope (1.013 ± 0.084) against the slope of 1.0 was found to be not significantly different from 1.0 (p = 0.132). Subsequently, the next data point on the V-slope was added and again tested against 1.0; the mean slope was 1.056 ± 0.090, significantly greater than 1.0 (p < 0.001). As the procedure was further repeated after sequentially adding the next data point, the mean S1 slope increased progressively. Furthermore, the individual linear regression line through the S1 data points varied depending on the number of data points included; a long S1 consisting of more data points appeared to converge to 1.0, whereas a short S1 with a few data points tended to diverge. However, the mean of all S1 linear regression lines (95 cases in total) was not significantly different from 1.0 (Additional file 3: Figure S3(c)).

The effect of the three different protocols (15, 25, and 50 W⋅min⁻¹) on RtShift (mL⋅min⁻¹ VO₂) was not statistically significant (15 W⋅min⁻¹⋅, 132.2 ± 52.5; 25 W⋅min⁻¹, 137.8 ± 48.5; 50 W⋅min⁻¹, 136.1 ± 49.3; p = 0.946).

Non-parametric statistical tests yielded concordant results with the parametric tests. The detailed results are presented in the additional file (Additional file 4).

During steady-state exercise, CO₂ storage in the tissue occurs at the start and disappears after 1–2 min, when presumably the storage space has been filled. This has been observed since the 1960s [4‐7]. In addition, the literature acknowledges that, even during incremental or ramp exercise testing, there is an artifact due to this phenomenon at the beginning of exercise and this segment of V-slope is to be excluded when VAT is determined [8, 11]. What has not been clearly pointed out is that during ramp exercise, this CO₂ tissue storage effect never disappears and persists at least until VAT begins (Fig. 4). Because of its nature, ramp exercise never achieves a steady state. From the observed persistence of CO₂ storage, it may be reasoned that the CO₂ storage space also increases and its rate of increase remains constant as the work rate increases. We hypothesize that, with the constantly increasing work rate, new groups of muscle fibers are recruited, resulting in a steady increase in new CO₂ storage space in active muscle.

CO₂ storage was initially measured by using hyperventilation and/or rebreathing methods [4‐6]. Later, CO₂ storage was calculated as the difference between the measured CO₂ and the predicted CO₂, based on the premise of a fixed respiratory quotient during sublactate-threshold exercise [12‐14]. Estimates from the hyperventilation methods, with the apparent inclusion of an HCO₃ ⁻ diffusing space in the whole body, were much larger than those from the rebreathing methods. The former measure of CO₂ storage was presumed to result only from tissue with a high metabolic rate, such as active muscle [14]. There is a possibility that the phenomenon of RtShift may be chiefly metabolic in origin, such as a shift to fat utilization as energy source, instead of due to CO₂ storage. However, we believe that this is highly unlikely because the RtShift occurs very early at the outset of exercise, whereas a major shift in the energy source to fat takes much longer to manifest, such as 20 min of steady-state exercise [15]. With ramp exercise too, there is no reason to believe that the energy source change to fat occurs so early, although no such basic study has ever been done during ramp protocols. Moreover and most important, the RtShift disappears (V-slope turns leftward) in 2–3 min while the work rate is being maintained at the same level (steady state). We assumed, on the basis of visual observations, that the slope of S1 of V-slope is approximately 1.0. However, the literature suggests that the RQ (R during steady-state exercise) increases by 0.05–0.1 from the resting value to moderate work rates [16]. This magnitude of RQ change is too small compared with R of either RtShift (far below 1.0) or S2 (far above 1.0). Therefore, we do not believe that the hypothesis of the slope of S1 being exactly 1.0 is an absolute requisite in the practical application of VAT and RtShift determination. Although there are no data on the energy utilization during ramp or rapid incremental testing, during work rate transition from rest to exercise, part of the energy fuel is estimated to come from phosphocreatine breakdown and glycolysis [17], both producing H⁺ and probably excess CO₂. Ramp exercise may be considered to consist of multiple work rate transitions; therefore, R may tend to increase slightly even during increments of light exercise intensity. Therefore, the observed slope of S1 of 1.0 (or its being parallel to R = 1 line) may be a result of a competing net effect of these factors (increasing slope of S1) and the RtShift (decreasing slope of S1). Wasserman states that the slope of S1 is slightly less than 1.0 [1].

The major finding of this research—that the size of the CO₂ storage space (RtShift) is associated with increased exercise tolerance—may, at first, seem surprising. However, the above hypothesis suggests that a greater mass of aerobic muscle fibers recruited for a given workload may be a contributing mechanism. Additionally, a greater level of carbonic anhydrase (CA) activity in muscle may be a factor. As it is now known that there are subtypes of CA expressed in muscle [18], the hydration of CO₂, and therefore the production and retention of HCO₃ ⁻ in muscle, may be facilitated by this mechanism. CA activity may increase as part of the overall increase in aerobic enzyme activity in muscle [19]. The only other study evaluating the relation between exercise tolerance and CO₂ storage is that of Chuang et al. [20], in which the authors demonstrated a significant positive correlation between O₂ deficit and CO₂ store in 12 relatively young healthy subjects. However, in their study, the direct relation between indices of exercise tolerance (VO₂max or VAT) and the size of the CO₂ store was not assessed.

The RtShift has several distinctive features that may be useful in the evaluation of exercise tolerance. Foremost among them is that RtShift does not require any assessor to determine its value. Second, its calculation is simple. Third, it has the additional advantage of an almost 100% determination rate. Fourth, RtShift, like VAT, does not require maximal exercise testing; it is mostly calculated from data points up to R = 1; therefore, it is unique in that it is primarily an aerobic phenomenon. Although it is significantly correlated with VO₂ peak and VAT, the correlation coefficient is only moderate (~0.7). Therefore, although it may not be used as a substitute for VAT, it may indicate that a yet-unidentified physiological factor that is absent in both VO₂ peak and VAT is involved. That these two parameters do not always move in parallel is obvious from the only moderate correlation efficient between them and the inspection of each case of V-slope (Additional file 2: Figure S2).

Multiple regression analysis was used to assess whether factors other than VAT accounted for the association between RtShift and VAT. It has been reported that hyperventilation before exercise caused a rightward shift of the V-slope [21, 22]. However, that was not a major factor in this study.

As shown in this study, RtShift may be quantitatively defined by more than one method. In this study, we chose a method of simple averaging. However, any other method may be devised as long as it can provide a valid estimate of RtShift during incremental exercise testing.

The substudy was performed because we were not sure how a ramp change of exercise protocol affected the size of RtShift. If the RtShift is dependent on the recruited muscle mass, then the steeper ramp may result in a greater RtShift. As shown in the Results section, ramp change did not result in a significant change in RtShift.

The RtShift data distribution was clearly not Normal, with a deviation to the lower values of RtShift. Log transformation of the data was performed; however, the statistical results of various analyses did not materially differ from those obtained with raw data. We also ran nonparametric tests with the concordant results with parametric tests. Therefore, we chose to present only nontransformed data for analysis, which has the benefit of visually corresponding to the shift on the V-slope graph. In addition, we believe that because of the relatively large size of our data set (n = 100), the central limit theorem applies [23].

A limitation of this study is that the exact physiological mechanism through which RtShift is related to exercise tolerance is, at present, only speculative. The exact biological basis of RtShift must be elucidated through basic science research. Another limitation is that we do not have an age- and sex-matched control population of sufficient size comparing RtShift between patients with cardiac disease and normal subjects. This issue must be systematically and prospectively addressed; in this study, we merely completed the process of quantifying RtShift.

In conclusion, during incremental CPX, there were varying degrees of rightward shift of the V-slope and they correlated significantly with VAT, suggesting that RtShift may be used as a completely objective measure of exercise tolerance. It is predominantly determined before the appearance of VAT. Its physiological meaning and clinical application requires further clarification.