Las Ciencias Sociales y el agro: un desglose temático

In considering paired metabolite correlation plots involving control patients one must expect variation as a result of differing glucose homeostasis, hormonal and metabolic adaptation178 and glycogen reserves. All of these factors may change with age178 (see section 4.1.4) as well as the general state of health of the subject.

4.1.1.1 Supply of free fatty acids to the mitochondria

As free fatty acids are release from adipose tissue in response to low blood carbohydrates one would expect a negative correlation between free fatty

acids,FFA, and glucose values, figure 3.02.. A poor correlation (cc=-0.4 table 3.03) may be partly caused by unevenly distributed glucose values but may also be a sign of a successful homeostatic mechanism. This is now explained.

In response to low blood glucose levels, fat metabolism is increasingly used as a

fuel source. If one were to plot activities of glycolysis and lipolysis against blood carbohydrate levels an initial relationship similar to that of region A in figure 4.01. would be seen.

Region A Region B. -\r Lipolytic Glycolytic or lipolytic activity Glycolytic Fast time

Fig.4.01. Theoretical plot of activities of glycolysis and lipolysis against blood carbohydrate levels.

However, as glucose levels fall lower, glucose conservation techniques (see section 1.2.2) become more pronounced. These have the effect of decreasing the rates of both the decline in glucose levels and the increase in fat metabolism (region B figure 4.01.).

In a healthy patient the level of FFA can be taken as an indicator of the rate of fat metabolism. Therefore, if the population of samples in this study extends into region B then the correlation of FFA(and to a lesser extent the %camitine esters and 3-hydroxybutyrate) against glucose will be diminished. Any such effect would be expected to show on fasting profiles for glucose and FFA. It is difficult to see any indicative pattern in Figures 3.19. and 3.20. although it should be noted that the patients used for these plots were only a proportion of all patients in the study. Therefore, it may be the case that some samples were taken outside region

A of figure 4.01.

Finally, the p value of no correlation for FFA against glucose controls is very low (p=0.0001, table 3.03) and, even when considering such a large sample size, is quite significant providing further evidence of correlation.

4.1.1.2 Transport into the mitochondrial matrix

As the CoA esters of the FFA reach the mitochondria they are converted to acyl carnitine to be transported across the inner mitochondrial membrane. Thus the level of esterified carnitine would be expected to increase with increasing FFA. As FFA increase in response to low blood glucose, carnitine esters should also be seen to increase. This is reflected in the plots of esterified carnitine against glucose (figure 3.05) and FFA (figure 3.14) which show very good linear correlations (CC=- 0.6 P=0.0003, and CC=0.6 P=0.0003 respectively, table 3.03).

The overall poor appearance of the other carnitine correlation plots is thought to be partially attributable to a smaller sample size. One negative result for esterified carnitine was recorded (Patient 103). Here the total and free values were very similar 18 pmol/1 and 22 pmol/1 respectively. A difference of 4 pmol/1 at approximately the 20 pmol/1 level lay within the limits of the variation of the assay (±2SD = ±2.4 - Quality control results 1989) and on this basis the negative carnitine result was assumed to be zero in the correlation plots. This value remains the lowest carnitine value whether it is negative or zero. Therefore, the correlation coefficients, being calculated on the ranks of the values, will remain unaffected.

Expressing carnitine esters as a percentage of the total carnitine value was seen to produce better correlations with the other metabolites than did the straight

forward esterified values. The reason for this is not clear but it may indicate some relationship between total and esterified values. For example, a diminished

supply of carnitine would limit esterification. However, this would not be

expected in normal healthy individuals. Further, if total carnitine was limiting to esterification the amount of free carnitine would be minimal. Low free carnitine values were not evident in these results.

4.1.1.3 Production of ketone bodies

Production of 3-hydroxybutyrate, 3HB, one of the ketone bodies, in association with increasing 13-oxidation rates is shown in correlations of this metabolite against glucose, FFA and percent carnitine esters, %CE (figures 3.06, 3.15 and 3.18

respectively).

Correlation of 3HB to %CE is extremely good (cc=0.8) possessing the highest correlation coefficients of all the plots. Interestingly the correlation coefficient for plots with metabolites involved in early stages of fatty acid metabolism decrease in accordance with how far one has travelled back along the pathway (see figure 4.03.). For example the correlation of 3HB against %CE is very high whilst that for 3HB against FFA is lower with 3HB against glucose lower still.

4.1.1.4 Correlation of intermediary metabolite values with lactate

There is no apparent correlation of lactate with any of the intermediary

metabolites (figures 3.01. and 3.07. to 3.11.). KJB Lamers only reported a very slight correlation with glucose (cc=0.34) and JP Bonnefont178 reported no significant difference between 15 and 24 hour fasting lactate values from children between the age of 1 and 12 months.

Lactate is both utilised by gluconeogenesis and produced by active skeletal muscle as described by the Cori cycle (figure 4.02.). The activity of the subjects in this study was not controlled and therefore, neither was their production of lactate. Further, gluconeogenesis is not solely reliant on the levels of lactate. Glycerol and amino acids also feed this pathway. Therefore, it is difficult to predict whether lactate will increase or decrease with length of fast. It maybe that lactate is too separated from fatty acid metabolism to show any clear relationships in plots against the other intermediary metabolites.

Glucose ■> Glucose Gluconeogenesis j Glycolysis 2P 6P -V Pyruvate /N Pyruvate Gluconeogenesis Lactate <- LactateV

LIVER BLOOD MUSCLE

Fig.4.02. The Cori cycle (P = ATP equivalents).

4.1.1.5 Effects of length of fast on intermediary metabolite levels

The negative correlation between glucose and length of fast (figure 3.19.) was as expected. The lactate profile is again poor probably for the same reasons

discussed earlier. The other intermediary metabolites FFA, %CE and 3HB all increase with fast (figures 3.20., 3.22., 3.23. respectively), as a result of rising demand for energy from non-glycolytic pathways. Total carnitine showed no change in response to fast and therefore because of the increase in %CE there was a necessary decrease in free carnitine (figure 3.21.). Interestingly, FFA/3HB

decreased as the length of fast increased (figure 3.24.). This indicates a growing rate of 3HB production relative to that for FFA. From figure 3.23. the relationship between 3HB and fasting time may be non-linear suggesting an increase in the rate of 3HB production with fast, within the first 25 hours at least. Such a

response may be connected to an increasing use of ketones as an energy source.

From figure 3.20, the rate of FFA increase may also rise during fast but to a lesser extent.

4.1.2 PAIRED METABOLITE CORRELATION PLOTS AND FASTING