Metabolism
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Bloating, flatulence and diarrhoea after drinking milk - but not yogurt or cheese
Abdul is a 25 year old doctor from Sudan; he came to London to study for a PhD 6 months ago and has suffered from diarrhoea and flatulence since arriving, although in Sudan he rarely suffered from gastrointestinal upsets of any kind. His diet is not very different from what he ate in Sudan, but he has been introduced to taking milk in his tea and coffee. At first he thought this was strange, because he remembered his grandfather telling him that when he was constipated he drank a glass of the milk that his grandmother was about to use to make yogurt.
Since his
diarrhoea was worrying him, he went to see a gastroenterologist, who sent him
for a lactose tolerance test. This involved coming in to outpatients in the
morning, not having eaten or drunk anything since the night before, drinking
a solution containing 50 g of lactose, then measuring the increase in blood
glucose over 3 hours. Within about half an hour of drinking the lactose solution
he suffered painful abdominal cramps, and "explosive" watery diarrhoea
with abdominal bloating and severe flatulence.
The results of the lactose tolerance test are shown below, with Abdul's results in red and the mean and standard deviation for 10 control subjects in blue.
What conclusions can you draw from this graph?
It is obvious that there is no rise in blood glucose at all after Abdul has drunk the lactose solution. This suggests that he lacks the enzyme lactase, and so cannot hydrolyse lactose to glucose and galactose.
Why do you think Abdul's blood glucose does not fall as low as in the control subjects after 3 hours?
The marked fall to below the initial (fasting) glucose concentration in the control subjects is the result of the insulin secreted in response to the rise in blood glucose caused by the lactose. Since Abdul had no increase in blood glucose after drinking the lactose solution, there was no increase in insulin secretion, and hence no resultant drop in blood glucose.
Why do you think Abdul suffered from abdominal pain, bloating, diarrhoea and flatulence after the test dose of lactose?
The unabsorbed lactose remains in the intestinal lumen and provides a substrate for bacterial fermentation in the colon, resulting in the production of a variety of compounds, including short-chain fatty acids (e.g. the 4-carbon compound butyrate, the 3-carbon compounds pyruvate and lactate, and the 2-carbon compound acetate), as well as carbon dioxide and small amounts of hydrogen and methane.
The formation
of these short-chain fatty acids results in a considerable increase in the osmolality
of the intestinal contents, drawing water into the intestinal lumen - hence
the watery diarrhoea. The bloating, pain and flatulence are the result of the
carbon dioxide and other gases produced by the bacteria.
Abdul was unfortunate in that he went to a gastroenterologist in a hospital where the original lactose tolerance test was used. A considerably less unpleasant test involves taking only a few grams of lactose, then measuring hydrogen exhaled in the breath.
How do you think the hydrogen produced by bacteria in the colon can be exhaled on the breath?
Hydrogen has (H2) is sparingly soluble in water, and also lipid soluble, so it will cross the intestinal mucosa into the bloodstream down a concentration gradient. In the lungs it will again cross the epithelium from the bloodstream into the alveoli down a concentration gradient.
Abdul's interest in his condition leads him to do some literature research - he wonders whether he is abnormal in lacking lactase as an adult. Certainly his mother tells him that as an infant he was breast fed with no problems. The outcome of his research is summarised in the table below:
| population group or country of study | % of adults lacking lactase |
| UK white | 4.7 |
| *northern Germany | 7.5 |
| Tuareg (nomads of the central Sahara) | 12.7 |
| *western Austria | 15.0 |
| *southern Germany | 23.0 |
| *eastern Austria | 25.0 |
| US black | 26.2 |
| Turkey | 71.2 |
| Sri Lanka | 72.5 |
| Italy | 75.0 |
| Greece | 75.0 |
| South African black | 78.0 |
| Japan | 89.0 |
| Singapore born Chinese | 92.4 |
| Canadian born Chinese | 97.9 |
| Papua New Guinea | 98.0 |
*The populations of northern and southern Germany are of different origin, as are the populations of western and eastern Austria.
Comparative studies show that most adult mammals lack lactase, although the newborn of most species have lactase activity and ar well able to tolerate milk until they are weaned. (The exceptions are the marine mammals, whose milk has a very high fat content and no carbohydrate; suckling marine mammals do not have intestinal lactase)
What conclusions can you draw from this information?
While most people of northern European origin seem to retain lactase into adult life, among people from tropical and subtropical regions it seems to be normal to lose lactase after childhood or adolescence, as happens with other mammals.
Until relatively recently, lack of lactase in adult life would not be noticeable among people in tropical and subtropical areas, since after weaning little milk is consumed. This is because until the widespread introduction of refrigeration, milk did not keep, and any milk that was consumed had been used to make yogurt. or cheese.
What is not clear is why persistence of lactase after adolescence is common among people of north European origin. One suggestion is that it provided a selective evolutionary advantage in countries where there was little sunlight exposure - milk is a significant source of vitamin D, as well as calcium - and in cold northern climates milk keeps reasonably well without refrigeration.
Now that he knows his problem, Abdul has stopped taking milk in tea and coffee, but he continues to consume yogurt, which he likes. Once he stopped consuming fresh milk his diarrhoea and abdominal discomfort ceased.
Why is it that Abdul (and other who lack lactase) can tolerate yogurt. perfectly well, but cannot tolerate milk?
Just as intestinal bacteria ferment lactose to short-chain fatty acids, so the bacteria used to make yogurt. (Lactobacillus spp) ferment lactose to lactic acid.
From his experience with lactose, Abdul has developed an interest in carbohydrate metabolism, and conducted a series of experiments to elucidate the pathway of glucose metabolism.
The metabolism of glucose in yeast
He began
his studies with the knowledge that under anaerobic conditions, yeast will metabolise
glucose (and other sugars) to form ethanol - the process of fermentation. Normally,
the carbon dioxide formed during the fermentation, being heavier than air, forms
a blanket over the fermentation vessel, so that it is effectively anaerobic.
If lactic acid bacteria, as used to make yogurt are incubated anaerobically with glucose, they form lactate, but no ethanol. Similarly, red blood cells incubated with glucose form lactate.
In his first experiments Abdul incubated, an amount of a yeast culture containing 10 mg of dry weight of cells in a final volume of 1 mL of a solution containing 300 mmol /L [14C-U]glucose in potassium citrate buffer at pH 3.5, in centre well vials under an atmosphere of nitrogen, at 20ºC.
(In
[14C-U]glucose the radioactivity is uniformly distributed among all 6 carbon
atoms)
The specific activity of the [14C]glucose was 0.1 µCi /mmol, so that each incubation contained 66,000 dpm of radioactive glucose.
After 1 hour, 1 mL of methoxymethylamine was injected through the seal into the outer compartment of the incubation vial, to trap carbon dioxide, and 0.5 mL of 1 mol /L perchloric acid was injected into the incubation mixture in the centre well, to kill the cells, precipitate proteins, and drive carbon dioxide out of the solution.
The flasks were shaken for a further 60 min, then:
The methoxymethylamine (containing carbon dioxide) was washed out with scintillator fluid and transferred to a scintillation counter vial.
The incubation mixture was washed out with water, and transferred to a micro still. It was evaporated to dryness, then:
The distillate (containing the ethanol formed in the incubation) was made up to 5 mL, and a 1 mL aliquot taken for liquid scintillation counting
The solid residue (containing the unreacted glucose) was dissolved in water, made up to 5 mL, and a 1 mL aliquot taken for liquid scintillation counting.
The results were as follows:
| dpm in sample | total dpm | µmol | |
| carbon dioxide - whole sample counted | 5500 |
? | ? |
| distillate (ethanol) - 1/5 of sample counted | 2200 |
? | ? |
| glucose remaining - 1/5 of sample counted | 9900 |
? | ? |
What conclusions can you draw from these results?
| dpm in sample | total dpm | µmol | |
| carbon dioxide - whole sample counted | 5500 |
5500 |
150 |
| distillate (ethanol) - 1/5 of sample counted | 2200 |
11100 |
150 |
| glucose remaining - 1/5 of sample counted | 9900 |
49500 |
225 |
This shows the stoichiometry of formation of 2 mol of ethanol plus 2 mol carbon dioxide from each mol of glucose. There is a net loss of 75 µmol of glucose, and formation of 150 µmol each of ethanol and carbon dioxide. Since label from [14C-U]glucose appears in both ethanol and carbon dioxide, it is obvious that both have been formed from glucose.
He repeated the experiments, but this time using glucose labelled in carbon 2, 3, 4, or 5, and the following results were obtained:
glucose labelled in |
||||
C-2 |
C-3 |
C-4 |
C-5 |
|
| carbon dioxide - whole sample counted | 0 |
16500 |
0 |
16500 |
| distillate (ethanol) - 1/5 of sample counted | 3300 |
0 |
3330 |
0 |
| glucose remaining - 1/5 of sample counted | 9900 |
9900 |
9900 |
9900 |
What conclusions can you draw from these results?
These results show that the disappearance of radioactive glucose is the same regardless of which positional isomer is used, and again if you work through the calculations there is consumption of 75 µmol of glucose. From the previous results, we know that this will lead to the formation of 150 µmol each of ethanol and carbon dioxide.
However, label only appears in carbon dioxide when the glucose is labelled in carbon 3 or 5, and none of the label from these two carbon atoms appears in ethanol.
Similarly, label only appears in ethanol when the glucose is labelled in carbons 2 or 4, and none of the label from these two carbons appears in carbon dioxide.
From these results, it is likely that glucose is split into two three-carbon units, each of which then undergoes decarboxylation to yield ethanol and carbon dioxide.
Fluoride and dental caries
It has been known for many years that people who live in areas where the drinking water contains about 1 ppm fluoride have very much less dental decay than those in areas where there is little fluoride in the water. This has led to the fluoridation of drinking water in many areas, and the widespread use of fluoride-containing toothpaste. The main effect of fluoride is incorporation into dental enamel, leading to increased resistance to attack by acid-forming oral bacteria, but, in addition to this there is evidence that fluoride inhibits the growth of acid-forming bacteria.
One of the main acid-forming bacterial of dental plaque is Streptococcus mutans; in studies in which this organism was incubated with 10 mmol /L glucose, the following results were obtained:
glucose consumed (µmol /h /mg protein) |
lactate formed (µmol /h /mg protein) |
|
| control | 6 ± 0.5 |
12 ± 1.1 |
| + 0.1 mmol /L sodium fluoride | 0 |
0 |
What conclusions can you draw from these results?
From the control incubations it is again apparent that 1 mol of glucose consumed leads to the formation of 2 mol of lactate.
Fluoride appears to inhibit the metabolism of glucose and the formation of lactate; presumably this accounts for its effect on the growth of the bacteria.
A problem with measurement of blood glucose
Abdul
was involved in the development of a new instrument for the automated measurement
of glucose in samples of whole blood. It consisted of a turntable containing
the samples to be analysed, which was rotated so that each sample in turn came
under a probe which withdrew 0.1 mL for reaction with the enzyme glucose oxidase,
and measurement of the colour developed in the presence of peroxidase and ABTS.
In one test of the instrument the instrument it was set up containing 6 samples of blood taken from volunteers before their breakfast, into heparinised tubes to prevent clotting, and was then left to run throughout the day, repeating the cycle of analyses each hour. The following results were obtained:
What conclusions can you draw from these results?
What should be done with blood samples that are to be used for determination of blood glucose?
The results show that the red cells, kept at room temperature, metabolise glucose, at a rate of about 0.8 mmol /L /hour; assuming that a “normal” blood sample contains about 160 g of haemoglobin /L, the rate of glucose utilization is about 21 µmol /g haemoglobin /hour.
Obviously, for determination of blood glucose, this metabolism must be inhibited.
The effect of fluoride on glucose utilization by S. mutans suggests
that fluoride might prove to be a suitable inhibitor, and the standard tubes
used for collection of blood for glucose determination do indeed contain fluoride.
The utilisation of glucose by red blood cells
Red cells were centrifuged out from freshly collected heparinised blood, and washed twice by resuspending gently in ice-cold phosphate buffered saline (0.1 mol/L NaCl, 0.05 mmol/L sodium phosphate at pH 7.4), and recentrifuging. They were then resuspended phosphate buffered saline to give a volume equal to the original blood volume.
Incubations were set up containing:
0.5 mL red cell suspension
0.25 mL of a solution of 40 mmol/L glucose
0.25 mL phosphate buffered saline
0.1 mL water or ADP, as shown in the table of results below
The samples were incubated for 30 min at 37ºC, when the reaction was stopped by the addition of trichloroacetic acid. After centrifugation to remove denatured protein, aliquots were used for the determination of glucose (using bacterial glucose oxidase, as above), lactate (using lactate dehydrogenase) and ATP (using the enzyme luciferase, which catalyses the hydrolysis of ATP to ADP and phosphate and emits light).
The results show the total amount of ADP added, and of glucose, lactate, and
ATP present at the end of the incubation in each tube (mean ± SD).
| ADP added (µmol) | glucose (µmol) |
lactate (µmol) |
ATP (µmol) |
| 0 | 9.55 ± 0.45 |
0.05 ± 0.01 |
0 |
| 2 | 8.55 ± 0.43 |
2.05 ± 0.12 |
2.45 ± 0.21 |
| 4 | 7.55 ± 0.35 |
4.05 ± 0.21 |
4.45 ± 0.31 |
| 6 | 6.55 ± 0.29 |
6.05 ± 0.33 |
6.45 ± 0.45 |
| 8 | 5.55± 0.19 |
8.05 ± 0.45 |
8.45 ± 0.52 |
| 10 | 4.55 ± 0.10 |
10.05 ± 0.50 |
10.45 ± 0.65 |
| unincubated control | 10 ± 0.36 |
0.05 ± 0.01 |
0.45 ± 0.02 |
What conclusions can you draw from these results?