2.1. Post-Exercise Effect
A representative 600 MHz
1H NMR femoral arterial plasma spectrum obtained from a subject at 70 min after both heavy resistance and intake of protein drink (calcium caseinate) is shown in
Figure 1.
Figure 1.
Representative 600 MHz 1H nuclear magnetic resonance (NMR) spectrum of a plasma sample 70 min after a bout of heavy resistance and intake of protein drink (calcium caseinate) (Red line). The difference between the 70 min post-exercise sample and the corresponding -70 min pre-exercise sample from the same individual is also shown (Blue line). Nomenclature: 1, lipid; 2, branched amino acids; 3, lactate; 4, alanine; 5, acetate; 6, N-acetyl glycoprotein; 7, glutamate/glutamine; 8, acetone; 9, acetoacetate; 10, citrate; 11, creatine; 12, cholines; 13, glucose; 14, mainly glucose region; 15, tyrosine; 16, histidine; 17, formate.
To determine the effect of exercise, PCA was carried out on all -70 and 70 min samples (
Figure 2). Principal component (PC) 6 in the score plot discriminates between the pre- and post-exercise samples. The loadings ascribe these changes to a decreased intensity of beta-hydroxybutyrate and choline and an increased intensity of amino acids: alanine, branched amino acids, glutamate, glutamine, histidine, lysine and tyrosine after exercise. No clear effects of exercise were observed on the signals from lipids.
Figure 2.
(a) Principal component analysis (PCA) score plot of all 1H NMR data from blood samples taken -70 (violet-diamond) and 70 (red-triangle) minutes after a bout of heavy resistance training. (b) PCA loadings for component 6. Nomenclature: 1, lipid; 2, beta-hydroxybutyrate; 3, choline; 4, branched amino acids; 5, alanine; 6, glutamate/glutamine; 7, lysine; 8, tyrosine; 16, histidine. The ellipse represents the Hotelling T2 with 95% confidence.
PCA was performed on the baseline-subtracted femoral arterial plasma samples for both whey protein and calcium caseinate. Both treatments showed a separation between the blood samples taken at 70 min after a bout of heavy resistance and the later time points (220 and 370 min; data not shown). To explore the biochemical differences,
Figure 3 shows an OPLS-DA model with one predicted and one orthogonal component (R2X = 0.42, R2Y = 0.82 and Q2 = 0.53) between time-points 70 and 220 min for the calcium caseinate treatment. The loadings for the predicted component indicates a higher intensity of beta-hydroxybutyrate and lipids at 220 min and a higher intensity of alanine, branched amino acids, creatine, glucose, glutamate/glutamine, histidine and tyrosine at 70 min after resistance exercise (
Figure 3(
b)). A similar OPLS-DA model for the intake of whey protein revealed similar dynamic changes in the post-exercise period (data not shown).
Figure 3.
(a) Orthogonal partial least squares regression-discriminate analysis (OPLS-DA) score plot of baseline subtracted 1H NMR data from blood samples taken 70 (red-triangle) and 220 (green-box) minutes after a bout of heavy resistance training and the intake of calcium caseinate drink. (b) PCA loadings for component 5. Nomenclature: 1, lipid; 2, beta-hydroxy butyrate; 3, branched amino acids; 4, alanine; 5, glutamate/glutamine; 6, creatine; 7, glucose; 8, tyrosine; 9, histidine. The ellipse represents the Hotelling T2 with 95% confidence.
2.2. Milk Protein Effect
To examine the differences between the different drinks ingested, PCA was carried out on all the baseline-subtracted femoral arterial plasma samples (
Figure 4). The control samples are found to separate along PC 1 from the whey and calcium caseinate samples. This discrimination can be ascribed to higher intensities of lipids and choline-containing compounds (choline, phosphocholine and glycerophosphocholine) after intake of milk proteins compared to the control. In addition, PCA models obtained at the individual time points for the milk protein samples discriminated between whey and calcium caseinate at 70 min after a bout of heavy resistance training. The loadings indicated higher lipid signal intensity from 0.81-0.85 ppm and 1.22-1.27 ppm (data not shown).
Figure 4.
(a) PCA score plot of all baseline subtracted plasma samples showing the different protein sources: orange-triangle, control; black-box, whey protein; grey-dot, calcium caseinate protein. (b) PCA loadings for component 1. Nomenclature: 1, lipid; 2, choline. The ellipse represents the Hotelling T2 with 95% confidence.
Subsequently, two OPLS-DA models with one predicted and one orthogonal component were performed to elucidate differences between the control samples and whey or calcium caseinate samples, respectively, at time point 70 min for baseline subtracted data (
Figure 5; water
vs. whey: R2X = 0.49, R2Y = 0.81 and Q2 = 0.59; water
vs. calcium caseinate: R2X = 0.44, R2Y = 0.79 and Q2 = 0.52). The loadings for the predictive components of both models show that intake of both whey and calcium caseinate proteins results in a higher intensity of branched amino acids, alanine, acetoacetate, creatine and tyrosine and a lower intensity of acetate, glycoproteins, citrate and glucose. However, after intake of calcium caseinate protein, all the lipid signals have positive loading values, whereas the lipid signals for the difference between non-caloric and whey drinks have both positive and negative values in the OPLS-DA loadings. The OPLS-DA model discriminating between control and whey (
Figure 5 (
b)) points to the higher intensity of the lipid signals from CH3 groups at 0.80-0.86 ppm and CH2 groups at 1.18-1.28 ppm. Conversely, the lipid signals at 0.87-0.90 ppm and 1.28-1.36 ppm have decreased intensity for whey samples compared to control samples.
Figure 5.
OPLS-DA score plots discriminating between control and whey (a) and calcium caseinate (b) at 70 min (baseline subtracted) after a bout of heavy resistance. Corresponding loading plot describing the component for OPLS-DA models of control vs. whey (c) and control vs. calcium caseinate (d). Nomenclature: 1, lipid; 2, branched amino acids; 3, alanine; 4, acetoacetate; 5, creatine; 6, tyrosine; 7, acetate; 8, N-acetyl glycoprotein; 9, citrate; 10, glucose. The ellipse represents the Hotelling T2 with 95% confidence.