3.2. Analysis of Blood Samples
The total protein in the blood of all groups was within the reference limits (64–83 g/L) and did not change significantly during all stages of observation (
Table 4), although there were certain variations in the fractional structure of proteins. To be more specific, the albumin fraction in the CG at each stage of the study was significantly higher compared with the initial values (8.2% and 15.5% (
p < 0.05), respectively). In TG 2, albumin increased 4.6% at the end of the final stage of the study (
p < 0.05), while in TG 1 it did not change.
The concentrations of α1- and α2-globulins reached the upper reference limit. The concentration of α1-globulins in the CG and TG 2 did not change throughout the entire observation period. In TG 1 this indicator significantly decreased (by 12.4% and by 14.6% (p < 0.05)) after the end of therapy with functional foods and at the end of the final phase of the study. At the same time, at the initial stage, the concentration of α1-globulin in TG 1 went beyond the upper reference limit.
The concentration of α2-globulins in the CG was initially above the norm. It decreased 13.9% (p < 0.05) by the time of the interim blood test. This decrease was 15.5% (p < 0.05) at the end of the final stage of the study. In TG 2, the fraction of this type of globulin also dropped significantly by 12.7% (p < 0.05) by the interim tests, but later on, it did not differ from the initial value. In TG 1, the concentration of α2-globulins did not change and remained stable.
What is interesting in
Table 3 is that in the CG and TG 2, there were no changes in the concentration of β-globulins during the observation period. In students of TG 1, by the end of the protein–plant FP therapy, the concentration of β-globulins increased 8% (
p < 0.05), but 30 days after the end of the intake, it went down to its initial value.
The concentration of γ-globulins in the blood serum of the CG at the second and third stages of observation were significantly lower than at the initial stage (18.7% and 19.0% (p < 0.05) respectively), and were below the norm. In TG 2, an 8.8% decrease (p < 0.05) below the norm in this protein was observed only after 45 days. The CG showed a 19.0% drop in γ-globulins whereas in TG 2 the decrease was not so large—8.8%—which is 2.1 times less than in the CG. In TG 1, after completing therapy with functional foods, the level of γ-globulins rose by 8.6% (p < 0.05) and remained high 45 days into observation (4.9% (p < 0.05)).
The dynamics of blood serum parameters showed that 100% of the CG subjects had an increased level of albumin at the time of the interim test, and one fifth of subjects exceeded the reference limit. By the end of the observation period, an increased level of albumin relative to the initial value was observed in 90% of the subjects. The initial α1- and α2-globulin levels were above the normal level in 50–60% of students. At the end of observation, high levels of these globulins were registered in 40% (α1) and 30% (α2) of the subjects. In addition, at the second and third stages of observation, the level of α1-globulins increased or remained the same relative to the initial value in 40–60% of students. An increased α2-globulin level at the second stage of the clinical trial was noted in 60% of the CG students and, by the end of observation, in 10% of students. A reduced level of β-globulins at the initial stage was registered in 20% of the students and, after 45 days, in 40%. A slight drop in β-globulin (though within the norm) relative to the baseline values occurred in 60–70% of the subjects. At the beginning of the observation, 10% of students had γ-globulin higher than the norm; 30% of subjects showed below-the-norm γ-globulin values. After 15 days, no subjects had high levels of γ-globulins, and 80% of subjects had much lower γ-globulin values compared with the initial ones. By the end of the observation, the number of students having low γ-globulin reached 50%, and its decrease from the initial value was noted in nine subjects out of ten.
In TG 1, the level of albumin did not exceed the reference limits, and the number of students having an increased level of albumin relative to the initial value did not exceed one third at each stage of the observation period. At the beginning of the observation, the level of α1-globulins was higher than normal in 45% of the subjects. At the interim stage, it was above the norm in 30% of the subjects, and by the end of observation, it was higher only in one out of ten students. At the 2nd and 3rd stages of observation, 60% and 80% of the students demonstrated a decrease in the level of α1-globulin relative to the initial value. The level of α2-globulins at the beginning of observation was higher than the norm in 60% of students and, at the end of observation, only in 20%. A drop relative to the initial individual data was registered in 65% and 60%, respectively, at stages 2 and 3. An increase in β-globulins was noted in 65% of the students by the end of the protein–plant FP intake, and one third of the subjects retained this level of content until the end of the final stage of the study. At the initial stage, the level of γ-globulin was reduced in 35% of the participants. By the end of the course of treatment with functional foods, the percentage of subjects with reduced γ-globulin levels was 25%, and after another 30 days (46th day) it reached 30%.
There were practically no differences in the albumin fraction between TG 2 and the CG. However, after 15 and 45 days, an increase in albumin was registered in 80% and 75% of the subjects, respectively. Increased α1-globulin levels were noted in 40% and 30% of the subjects. However, the level of α1-globulin decreased in 60% of the individuals in this group. No differences in α2-globulin levels were found in the TG 2 compared to the CG. At the same time, a 15–20% decrease in the level of β-globulins in relation to the initial values was registered in a smaller number of subjects. The levels of γ-globulins gradually rose in a larger number of subjects during the stages of observation, while a decrease was noted in a smaller number of subjects than in the CG.
Red blood cell counts at all stages of observation were within the normal range (
Table 5). From these data it can be seen that the number of erythrocytes, hemoglobin concentration, and hematocrit did not change significantly during the observation period. However, the mean erythrocyte volume considerably increased in each group. The mean erythrocyte hemoglobin level did not change in TG 1, while in TG 2 the mean erythrocyte hemoglobin decreased by 1.1% (
p < 0.05) at the end of the final stage of the study. The same response was noted in the CG.
The average hemoglobin level decreased markedly at the second and third stages of observation in all students. The production of erythrocytes increased in the TG 1 group by 2.5% (p < 0.05) after a course of treatment with functional foods; at the end of the observation period, it was 2.9% higher than the initial value (p < 0.05). TG 2 subjects also showed a 2.3% increase in erythrocyte production (p < 0.05), but at the end of the clinical trial, there was no significant deviation from the initial value. No such patterns were observed in the CG.
In TG 1, white blood cell count increased by 12.0% (
p < 0.05) by the end of the course and the level of content remained unchanged until the end of the final stage of the study (
Table 6). In TG 2 and CG, no relevant changes in the white blood cell count were registered.
These tests also showed that, during the period of observation, the number of lymphocytes increased. In TG 1 there was a considerable rise in the number of lymphocytes by 9.4% (p < 0.05), which continued up until the end of the final stage of the study (by 8.6% (p < 0.05). In TG 2 and CG, a significant increase of this parameter in comparison with the initial value was registered only at the end of observation—22.7% (TG 2, p < 0.05) and 24.9% (CG, p < 0.05). The relative level of lymphocytes (fraction of total leukocytes, %) in TG 1 at the second and third stages of observation was more significant than that at the initial stage, exceeding it by 14.3% (p < 0.05) and 36.7% (p < 0.05), respectively. In TG 2, a strong upward tendency was identified, reaching a 15.1% (p < 0.05) increase in the level of lymphocytes at the end of observation. There were no statistically significant differences in the CG data.
Neutrophils in TG 1 rose by 17.3% (p < 0.05) at the end of the final stage of the study within the normal limits of nutritional intervention. At the same time, at the end of the final stage of the study, this value changed slightly compared to the initial one (p < 0.05). The number of neutrophils during the stages of observation in the TG 2 and CG groups did not change significantly, remaining within normal limits. Their relative number in TG 1 and CG did not change during the stages of observation, and in TG 2 this parameter decreased by 9.4% (p < 0.05) by the end of observation.
In TG 1, there was a considerable 17.2% (p < 0.05) increase in the level of monocytes, basophils, and eosinophils at the end of the final stage of the study. The relative values of these blood-forming cells did not change during the stages of observation.
By the end of the course the leukocyte count increased 12.0% (
p < 0.05) in TG 1 and remained unchanged (
p < 0.05) until the end of the final stage of the study (
Table 6). In TG 2 and CG, no statistically relevant changes were registered. During the period of observation, there was an increase in the number of lymphocytes. In TG 1, a marked increase in the number of lymphocytes by 9.4% (
p < 0.05) was registered, which continued up to the end of the final stage of the study (8.6 % (
p < 0.05)). In TG 2 and CG, this parameter grew in comparison with the initial value, however, this growth was registered only at the end of the observation—22.7% (TG 2,
p < 0.05) and 24.9% (CG,
p < 0.05), respectively.
The relative count of lymphocytes (fraction of total leukocytes, %) in TG 1 at the second and third stages of observation was 14.3% (p < 0.05) and 36.7% (p < 0.05) higher than at the initial stage, respectively. TG 2 showed a 15.1% (p < 0.05) increase by the end of observation. No statistically relevant changes were registered in the CG.
Neutrophils in TG 1 rose by 17.3% (p < 0.05) regarding the reference values by the end of the period of the nutritional intervention. However, until the end of the final stage of the study, this value did not differ significantly from the baseline (p < 0.05). The number of neutrophils during the stages of observation in the TG 2 and CG groups did not change, remaining within the norm. Their relative number in the TG 1 and CG was stable during all stages of observation, whereas this rate in the TG 2 group decreased slightly by 9.4% (p < 0.05) by the end of the final phase of the study.
TG 1 showed an upward trend in the combination count of monocytes, basophils, and eosinophils, which at the end of the final phase of the study was 17.2% (p < 0.05). The relative value of these blood-forming cells did not change during the stages of observation.
The highest platelet counts ((243.9 ± 8.50)× 10
9 cells/L) were observed for group 3 after the protein–vegetable product TG 1 diet (
Table 7). The highest mean platelet volume was observed in control group 1 (10.82 ± 0.38 fl).
In the CG, serum immunoglobulin A was initially at the lower limit of the norm, decreasing 37.5% (
p < 0.05) at the end of the final stage of the study. At each stage of observation, it was lower than the norm in 90.0% of CG subjects (
Table 8). The mean IgM index in the CG was initially within the normal range. After 15 days, the mean value was dramatically higher (80.4% (
p < 0.05)) than the initial value. In the CG it rose in 100.0% of subjects, and in 30.0% of students its value reached the upper limit of the norm. After 45 days, the mean IgM value was 54.0% higher than that of the baseline (
p < 0.05). IgG at the baseline and at the observation stages was within its reference values.
In TG 1 and TG 2, mean IgA values were within a normal range and did not change much during the stages of observation. IgM was within the norm in all TG 1 subjects. However, it increased 43.3% (p < 0.05) after 45 days of observation, whereas TG 2 showed a 32.2% (p < 0.05) increase of this parameter after 15 days of observation and a 68.2% (p < 0.05) increase at the end of the observation period.
In TG 1 the content of IgG increased compared to reference limits by 43.3% (p < 0.05) at the end of the final stage of the study, while TG 2 demonstrated a 34.8% (p < 0.05) increase in IgG on the 15th day of observation and a 38.5% (p < 0.05) increase at the end of observation.
The testosterone level in the CG during all the stages of observation was within the normal range (
Table 9). However, after only 15 days of the study there was a substantial drop in its level by 29.6% (
p < 0.05). It continued to fall and, after 45 days, the decrease was 32.8% (
p <0.05). It is noteworthy that at each stage of observation 100% of the subjects showed a downward tendency in the level of testosterone. In TG 1, the level of the hormone decreased only towards the end of the observation period and the decrease was not significant. In TG 1, 40.0% of subjects had an increase in testosterone by the end of the FP course, and in 25.0% of subjects, this increase persisted for one month after the end of the FP course. A minor drop in testosterone was also observed in TG 2 students. It decreased in all the subjects but was still within the normal range.
Blood cortisol in CG subjects was above the reference limits in 100.0% of subjects during the first and second examinations. Individually, it was initially high in 50.0% of students and, after 15 days, in 70.0%. After another month, its mean value was within the upper limit of the norm, but 60.0% of the individuals examined had a cortisol level higher than the upper reference value. In TG 1 and TG 2, blood cortisol was within normal limits during the whole period of observation. However, at the initial stage, cortisol was above the norm in 40.0% of TG 1 subjects and, after 15 days, in 50.0%; in TG 2 it was above the norm in 40.0% and 60.0% of students, respectively. After 45 days, only 25.0% of TG 1 subjects and 40.0% of TG 2 subjects had an increased cortisol level.
Vitamin A was above the reference limits in subjects of all groups (
Table 10)—no individuals with low levels of the vitamin were found. By the end of the FP course, a considerable increase in the mean values relative to the baseline data was observed; in TG 1 it was 21.3% (
p < 0.05) and in TG 2—15.5% (
p < 0.05). After another 30 days, each group retained these high levels. However, in TG 1 the concentration of vitamin A was 23.6% (
p < 0.05) higher than the baseline, and in TG 2 a slight drop was observed, but the value was still 10.9% (
p < 0.05) higher than the baseline. CG subjects showed no changes.
Fifteen days into observation, in the CG vitamin E was 12.5% (p < 0.05) lower than the baseline value and remained stable until the end of observation. Initially, a decreased vitamin E concentration was found in 40.0% of the subjects and, after 15 days of observation, in 80.0%, remaining the same in 80.0% of subjects up to the end of observation. Initially, in TG 1, vitamin E concentration was also below the norm, but after a course of taking functional foods and at the end of the final stage of the study it was within the normal range, having grown 10.7% (p < 0.05) and 16.9% (p < 0.05), respectively. At the beginning of the clinical trial, only 40.0% of subjects had normal levels of this vitamin. During the FP course, 65.0% of subjects reached the norm, and at the end of the observation 70.0% of individuals had normal levels of this vitamin. Other subjects also showed positive dynamics; however, the concentration of vitamin E did not reach the normal values. In TG 2, the initial mean value of this vitamin was well within the normal range (in 55.0% of subjects). During the FP course, the mean value did not change significantly, although the percentage of individuals having the normal level of vitamin E rose to 65.0%, and an increase in serum vitamin E was noted in 45.0% of subjects. By the end of the observation, normal levels of this vitamin were registered in 80.0% of the TG 2 students, with an increase of 19.5% (p < 0.05).
The concentration of vitamin B1 in all groups was above the reference limits. Vitamin B1 levels in the CG dwindled during all periods of observation, with a 17.0% (p < 0.05) decrease after the course of FP intake and a 10.7% (p < 0.05) decrease below the baseline values at the end of the observation. In TG 1 no such dynamic was noted. However, in TG 2, the concentration of vitamin B1 fell by 18.4% (p < 0.05) by the end of the final stage of the study.
The tests showed that, initially, most subjects of all groups had vitamin B2 deficiency. After the FP intake, TG 1 subjects showed a rise of 13.5% (p < 0.05) and, by the end of the final stage of the study, 28.3% (p < 0.05), though still not reaching the normal value. A rise in the concentration of vitamin B2 by stages of observation was noted in 25.0% and 75.0% of subjects, respectively. A slight rise of 7.0% (p < 0.05) in the mean value of the vitamin was also noted in TG 2 by the end of observation. The increase in the concentration of this vitamin was registered in 35.0% (21 days) and in 60.0% (37 days) of students.
When assessing blood mineral levels, we found that the concentration of iron in the CG and TG 2 did not change much at any stage of the observation and remained within the normal range (
Table 11). In TG 1 a significant 15.2% rise in iron (
p < 0.05) was observed at the end of the FP treatment, which continued up to at the end of the final stage of the study (a 20.8% increase (
p < 0.05)). At the initial stage, 35.0% of the subjects had iron levels below the norm, but at the end of the study and at the end of the observation period, just 5.0% of students still retained their low initial value. In TG 2, at the beginning of the FP course 15.0% of the subjects had their iron level below the norm, but at the end of the FP intake only 5.0% had iron deficiency. After 45 days, 100.0% of subjects had normal iron values.
In the CG, magnesium was within the norm during all periods of observation, but after 15 days of adaptation, its concentration fell to much lower than the initial value, with a massive drop of 22.6% (p < 0.05), and after 45 days of adaptation it was 12.8% lower (p < 0.05). A gradual decrease by stage of observation relative to the initial values was noted in 100.0% and 80.0% of the observation groups. In TG 1, at the beginning of the study, magnesium was generally below the norm, and 75.0% of subjects showed considerable deviations. At the end of the FP course, there was a 6.3% increase (p <0.05) in magnesium, and the percentage of individuals with low or decreased magnesium levels went down to 40.0%. By the end of observation, 100.0% of subjects had adequate magnesium concentrations and its average value increased by 34.9% (p < 0.05). In TG 2, magnesium was within the norm, but after 15 days its concentration decreased 14.3% (p < 0.05), and, by the end of the final stage of the study, it was the same as the baseline value. At the beginning of the study, 25.0% of the individuals had magnesium levels lower than the norm. Later on, during all stages of observation, it was within normal limits in all those tested.
Initially, the concentration of phosphorus was within the reference values. In the CG, after 15 days of the study, there was a 10.3% (p < 0.05) decrease in the concentration of the mineral. By the end of the observation, it did not differ from the initial value. In TG 1, a remarkable 16.1% (p < 0.05) increase in phosphorus levels was observed after the FP intake, which continued up to the end of the final stage of the study (increase by 20.5% (p < 0.05)). In TG 2, the concentration of phosphorus did not change during the stages of observation.
The content of potassium, calcium, sodium, and chlorine in both groups of students did not differ significantly from the control values. No statistically significant changes in the concentrations of these minerals were observed (p > 0.05) in both groups. However, sodium, which in the CG initially and after 15 days of observation was within the norm, exceeded it by the end of the observation period. There was an upward tendency in the values of this mineral at all stages of observation. At the beginning, it was higher than the norm in 30.0% of subjects, and by the end of observation it was up in 70.0% of subjects. In TG 1, sodium was within the normal range. Initially, it was higher than the norm in 30.0% of subjects, by the end of the course only in 5.0%, and at the end of the observation period just in 10.0%. The sodium level in TG 2 did not differ much at different stages of observation and was within the normal range. Initially, it was higher than the norm in 15.0% of students, after 15 days in 5.0%, and after 45 days in 30.0% of those observed.
In the CG, at the beginning and after 15 days of observation, chlorine was within the norm, and after 45 days, the mean value was above the norm. By the second stage of observation, there was an increase in the chlorine concentration relative to the initial value in 60.0% of the subjects and, by the end of the final stage of the study, in 90.0%. In TG 1 there was a 1.3% increase in the level of chlorine by the end of the FP intake. However, by the end of the final stage of the study, it was significantly lower than the initial value (by 1.7% but still within the norm). In 100.0% of subjects, it remained normal during all stages. In TG 2, by the end of observation the concentration of chlorine was 6.5% higher than the baseline value. No individuals exceeded the norm during the stages.