Principales componentes de la dinámica familiar

As discussed in section 2.3.2, impaired gallbladder motor function is likely to play an important role in the formation of cholesterol gallstones. Improving gallbladder emptying has been shown to prevent gallstone formation in animals. Gallbladder “sludge” is a term coined for the echogenic layer sometimes found in the gallbladder at ultrasonography (Conrad et al, 1979). It is composed o f cholesterol crystals and bilirubin granules embedded in a matrix of mucus gel. Gallbladder sludge is a precursor of gallstones in some patients and, like gallstones, can be prevented by improving gallbladder emptying (Sitzmann et al, 1990). In patients receiving continuous or intermittent total intravenous nutrition (IVN), meal-induced stimulation o f gallbladder emptying is absent (Cano et al, 1986) with the consequence that sludge and gallstones form in up to 60% and 25% of patients respectively (Messing et al, 1984).

The rate of delivery of nutrients during IVN is relatively slow and the aim o f this study was to determine if a rapid intravenous infusion of amino acids stimulates release of CCK and gallbladder contraction.

10.1 Subjects

A randomised double-blind study was performed in eight healthy volunteers, aged 24 - 36 years (four men). All were non-smokers and within the normal weight range. None were taking any medication at the time of the study.

10.2 Methods

Subjects received 250 ml sodium chloride (300 mmol/1) over 10 or 30 min, or one of four different amino acid regimens administered intravenously after an overnight fast of at least 10 hours. The amino acid infusion used was Synthamin 14 without electrolytes (Baxter Healthcare, Norfolk, UK) (table 6). The four regimens of amino acids used are listed in table 7.

Essential amino acids Non-essential amino acids L-Leucine 6.20 L-Alanine 17.60 L-Isoleucine 5.10 L-Arginine 9.78 L-Lysine 4.93 L-Proline 5.78 L-Valine 4.93 L-Serine 4.25 L-Phenylalanine 4.76 L-Tyrosine 0.34

L-Histidine 4.08 Aminoacetic acid &76

L-Threonine 3.57

L-Methionine 3.40

L-Tryptophan 1.53

Table 6

Aminoacid content o f Synthamin 14. Values are in g/l

Infusion Total dose of amino acids (g) Rate (g/min)

1 250 ml over 30 min 2L2 0.7

2 250 ml over 10 min 2T2 2.1

3 125 ml over 5 min 10.6 2.1

4 50 ml over 5 min 4.2 0.8

Table 7

Gallbladder ultrasound scans were performed by Dr J Healey (Senior Registrar in the Department of Radiology, St Bartholomew’s Hospital). Gallbladder volumes were determined using the ellipsoid method in which the volume o f the gallbladder is approximated to that of an ellipsoid by the formula tt/ 6 x length x width x height. This

method has previously been shown to be a valid and accurate method o f determining gallbladder volume by ultrasound (Dodds et al, 1985). Measurements o f gallbladder length, width and height were made before the amino acid/saline infusion, and at 5 minute intervals for Ih.

Blood samples were taken via a 21-gauge butterfly cannula placed in a forearm vein at the beginning of each experiment. Samples were taken before the infusion and after 10, 20, 30, 45, and 60 min. Blood was collected as described in section 6.5.2.1 and plasma stored at -70^C until assayed for CCK using the bioassay.

10.2.1 Statistical analysis

The data is expressed as means ± SEM. Comparisons between groups were made with the Friedman test; Wilcoxon’s signed rank test with Bonferroni correction was used for multiple comparisons. Absolute gallbladder volumes, rather than percentage changes, were used for comparisons.

10.3 Results

The maximal percentage gallbladder emptying during each o f the infusions was determined using the formula:

(1-RV/FV) X 100, where RV = residual volume and FV = fasting volume

In control experiments it was shown that 250 ml of 300 mmol/1 sodium chloride administered intravenously over 10 or 30 min had no effect on gallbladder volume or plasma CCK concentrations.

All intravenous amino acid regimens produced significant gallbladder emptying except 50 ml over 5 min. The maximal percentage gallbladder emptying with 50 ml in 5 min was significantly less (p < 0.01) than the other three regimens (figure 28). 125 ml in 5 min was the lowest amino acid load which induced a significant emptying o f the gallbladder. With this dose, the mean gallbladder emptying was greater than 60% in 6 o f the 8 subjects. &

t

I

Maximal gallbladder emptying with the four regimens of intravenous amino acids. Regimen 4 (50 ml in 5 min) was significantly less effective (p < 0.01) that the other three regimens.

Figure 29 shows the time course of gallbladder emptying with each of the amino acid regimens. With infusion of 125 ml in 5 min the gallbladder reached its minimal volume at 30 min and gradually started to refill by 40 min. This is similar to the pattern seen after oral administration of a liquid fatty meal. In contrast, the higher doses of amino acids produced prolonged contraction of the gallbladder; minimal gallbladder volume was reached at 35 and 45 min with infusion of 250 ml over 10 min and 250 ml over 30 min respectively and the volume had not increased by 60 min with either infusion.

(U S 'o > Ul <u T3 T3_CT3 O 30 20 . . + . . 5 0 m l/5 m in * 125 ml/5 mi n o 2 5 0 m l /1 0 min 2 5 0 m l /3 0 min 10 0 50 60 10 20 30 40 0 Time (minutes) Figure 29

Peak plasma CCK concentrations were significantly {P <0.01) higher than basal values in all four amino acid regimens. Peak CCK did not differ significantly during the three longer infusions, but was significantly {P <0.02) lower during the infusion of 50 ml in 5 min (figure 30). Integrated CCK concentration, expressed as area under the curve (pmol/1/60 min), was significantly lower {P < 0.02) with the regimen of 50 ml over 5 min (82.4 ± 6.3) than with 125 ml over 5 min (192.9 ± 20.6), 250 ml over 30 min (211.3 ± 32.3) or 250 ml over 10 min (235.6 ± 16.3). o S 3 y! U U 10 ..♦ ..5 0 ml/5 min ♦ 125 ml/5 min _ e _ 2 5 0 ml/10 min - n- 250 ml/30 min 8 6 4 2 0 10 0 10 20 30 40 50 60 Time (minutes) Figure 30

Plasma cholecystokinin concentrations in response to intravenous amino acid infusion in 8 healthy subjects. Values are the mean ± SEM

10.4 Discussion

The standard rate for infusion of IVN is 2,500 ml over 24 hours delivering approximately 106 g o f amino acids at an infusion rate o f 73.6 mg per minute. In this study, 125 ml (10.6 g) of amino acids infused over 5 min (2.1 g/min, regimen 3) was the smallest dose to produce significant gallbladder emptying. It was found that the rate o f amino acid infusion as well as the amount given determine the extent o f gallbladder contraction. At an infusion rate of 0.7 - 0.8 g/min there was contraction with 21.2 g (regimen 1) but no contraction with 4.2 g (regimen 4). A higher infusion rate o f 2.1 g/min, however, produced contraction with a load of only 10.6 g (regimen 3); this infusion rate is almost 30 times that obtained during standard IVN. Although in this study gallbladder contraction was not measured during standard IVN infusion rate, other investigators have shown no change in gallbladder volume (Nealon et al, 1990). In the same study Nealon and co-workers measured venous plasma concentrations of amino acids during intravenous infusion of amino acids and after ingestion o f a mixed meal containing 25 g of protein. The amino acid composition of the meal was not known and so cannot be directly compared with the intravenous amino acids. However, peak plasma concentrations of L-tryptophan, L-phenylalanine and L-leucine were comparable after meal ingestion and after infusion of 0.3 g/min amino acids for 60 min (total dose 18 g). This infusion rate is lower than for any of the four regimens used in our study. Therefore it is likely in the present study that plasma amino acid concentrations were well above those seen after ingestion of a mixed meal.

In this study four subjects experienced mild side effects during infusion of 250 ml over 10 min but not with lower infusion rates. The side effects were of transient flushing in two subjects and “light headedness”, without a change of pulse or blood pressure, in a further two subjects. Thus, rapid infusion of amino acids appears to be a safe and effective method of causing gallbladder contraction in patients undergoing IVN who are at risk of sludge and gallstone formation.

Release of CCK by intravenous amino acids would suggest that CCK is the main mechanism involved in amino acid-induced gallbladder contraction. Substance P,

motilin, gastrin releasing peptide and prostaglandins have all been shown to cause gallbladder contraction however the physiological role of these substances has not yet been determined. O f these, only release of motilin, is altered by administration of intravenous nutrients. Furthermore, it is intravenous fat and not amino acids which stimulates release o f motilin (Christofides et al, 1979). Thus it would seem unlikely that a hormone, other than CCK, is responsible for gallbladder contraction during amino acid infusion.

Two other studies have examined the effect of intravenous amino acids on CCK release. Nealon and co-workers (1990) showed that an intravenous infusion o f an amino acid mixture increased plasma CCK concentrations and caused gallbladder contraction. Infusion of 240 mg/kg/h (0.28 g/min for a 70 kg person) for 1 h induced significant gallbladder contraction to about 70% of fasting volumes and significantly increased plasma CCK concentrations. However, both fasting and stimulated plasma CCK concentrations were much higher than are usually reported and it is not clear from the methodology as to the extent of validation of the radioimmunoassay which was used.

In a later study performed at the same time as our study. De Boer and co-workers found that an intravenous infusion of an amino acid mixture at a rate of 250 mg/kg/h (i.e. for a 70 kg person, 17.5 g/h [0.29 g/min]) for 2 hours (total dose 35 g for a 70 kg person) did not increase plasma CCK concentrations but induced significant gallbladder contraction to 45% of the fasting volume (de Boer et al, 1993). In contrast, we did not find that a higher rate of infusion of 0.8 g/min produced gallbladder contraction, however the total dose administered was only 4.2 g compared with approximately 35 g in the study discussed above. Interestingly in the study by de Boer et al, gallbladder contraction in response to amino acid administration was completely blocked by the CCK^^ receptor antagonist, loxiglumide. This would suggest that CCK was responsible for gallbladder contraction although increased plasma concentrations could not be detected. It is possible that CCK mediated gallbladder contraction in this situation is a local effect; that is CCK is released fi*om gut endocrine cells or enteric nerves by circulating amino acids and is transmitted by neural pathways to the gallbladder. A higher infusion rate of

amino acids, as in our study, may stimulate increased release o f CCK which can then be detected in the peripheral blood.

Ingestion o f amino acids in human subjects causes release of CCK into the portal blood stream. It is not clear how luminal amino acids cause release of CCK but as discussed in section 1.5.4, experiments with isolated canine jejunal cells suggest a direct interaction with the CCK cell (Barber et al, 1986; Koop and Buchan, 1992). Release of CCK by intravenous amino acids suggests that they may interact with the basolateral side of the endocrine cell as well as the luminal side to cause release. Certainly it has been shown that there is transport of some amino acids, including phenylalanine, across the basolateral membrane into the enterocytes (Ghishan et al, 1989).

11. CCK RELEASE BY PHENYLALANINE AND ITS EFFECT ON