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.

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.

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.

This scenario is based on a case report / personal paper by a Sudanese doctor who suffered in this way after he came to London, and described his lactose tolerance test as one of the most impressive events of his life. It was published some years ago in BMJ or Lancet - I cannot find the reference - if anyone can find it for me I will be most grateful.

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.

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:

*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)

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.

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.

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:

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:

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:

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).

When no ADP is added little or no glucose is metabolised and little or no ATP is formed. This suggest that the utilisation of glucose is linked to the formation of ATP from ADP and phosphate.

For each 2 mol of ADP added, 1 mol of glucose is utilised, leading to the formation of 2 mol of lactate. At the same time 2 mol of ATP are formed.

Comparing the unincubated control and the sample incubated without ADP suggests that there is normally a small amount of ATP in erythrocytes, and this can lead to disappearance of 1 mol of glucose per mol of ATP. Very little of this appears as lactate - in fact, the amount of lactate formed is about the same as the amount of ADP that would normally be present in erythrocytes.

When the experiment was repeated using blood that had been allowed to stand at room temperature for 6 hours, there was no utilisation of glucose, and no formation of lactate, regardless of the amount of ADP added. None of these samples contained any detectable ATP.

However, when 1 µmol of ATP was added, glucose was utilised and lactate was formed, again increasing with increasing concentrations of ADP added.

The incubation of fresh red cells without any added ADP suggested that there is a need for ATP for glucose metabolism. The lack of any glucose utilization in the cells that had been allowed to stand at room temperature for 6 hours is presumably because during this time the endogenous ATP had been used in ion transport.

The restoration of glucose utilization by the addition of ATP shows that the enzymes had retained activity, and provides further evidence that there is a need for ATP, as well as ADP, for glucose metabolism.

Glucose uptake into red blood cells

If you want to study the uptake of glucose into red blood cells then the easiest way is to use 2-deoxyglucose, which is taken up by the cells, but is not metabolised. Using [14C]2-deoxyglucose means that it is easy to measure how much has been accumulated in the cells by washing them and determining radioactivity in an aliquot.

Incubations were set up containing:
0.5 mL fresh red cell suspension
0.25 mL phosphate buffered saline or a solution of 40 mmol/L glucose
0.25 mL phosphate buffered saline or 80 mmol/L [14C]2-deoxyglucose
0.1 mL of a solution of 100 mmol /L ADP
0.1 mL of buffer or a solution of 1 mmol /L sodium fluoride

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), ATP (using the enzyme luciferase, which catalyses the hydrolysis of ATP to ADP and phosphate and emits light) and 2-deoxyglucose by measurement of radioactivity.

The results show the total amount of glucose, lactate, ATP and 2-deoxyglucose present at the end of the incubation in each tube (mean ± SD).

ND = not detectable

The first row confirms the previous results. Approximately 2 mol of lactate and 2 mol of ATP are formed per mol of glucose utilised.

The incubation with 2-deoxyglucose shows only a very small amount accumulated; any glucose that was present in the cells initially has been utilised to form lactate, and any ATP that was initially present has also been utilised.

With both glucose and 2-deoxyglucose present almost all of the added glucose has been utilised, forming lactate (again approximately 2 mol of lactate formed per glucose utilised), but now there is no detectable ATP. Almost all of the 2-deoxyglucose has been accumulated in the cells. This must be active transport, against a concentration gradient (see the exercise on poisoned by unripe ackee fruit).

Assuming that 2 mol of ATP has been formed from each mol of glucose utilised, this suggests that each mol of 2-deoxyglucose accumulated has been at the expense of 1 mol of ATP consumed.

The results in the presence of fluoride, which inhibits glucose metabolism, show that metabolism of glucose is essential for the production of ATP to permit the accumulation of 2-deoxyglucose.

Thin layer chromatography of an extract of the red cells incubated with glucose and [14C]2-deoxyglucose shows that almost all of the radioactivity is present as 2-deoxyglucose 6-phosphate, not free 2-deoxyglucose.

It is likely that deoxyglucose can enter the cells freely, and is phosphorylated to 2-deoxyglucose 6-phosphate which cannot cross the cell membrane.

You could test this by incubating cells with 2-deoxyglucose 6-phosphate and glucose. If this hypothesis is correct then no 2-deoxyglucose 6-phosphate will accumulate in the cells.

Further studies show that the same enzyme, hexokinase, catalyses the phosphorylation of glucose to glucose 6-phosphate and of 2-deoxyglucose to 2-deoxyglucose 6-phosphate. It also catalyses the phosphorylation of other 6-carbon sugars (hexoses).

The effect of benzoate on glucose metabolism in yeast

Benzoic acid is commonly used as a food preservative, to prevent the growth of yeasts and fungi in moderately acidic foods (e.g. fruit juice, at about pH 3.0 - 3.5). It is a weak acid, with a pKa = 4.2.

At any pH below its pKa, any group that can accept a proton will do so, so the benzoic acid will be protonated.

Protonated benzoic acid is lipid soluble, and will readily cross cell membranes; intracellularly it dissociates, and the benzoate ion does not cross cell membranes. This means that cells incubated in moderately acidic media can accumulate as much as a 10-fold higher concentration of benzoate intracellularly than is present in the medium. At high intracellular concentrations, benzoate is toxic to cells such as yeast, which lack any mechanism for its onward metabolism.

The yeast Zygosaccharomyces bailii is peculiarly resistant to benzoate, and can grow in media containing up to 4 - 6 mmol/L, a concentration that prevents the growth of most yeasts. This means that fruit juice, etc, preserved with normal concentrations of benzoate will still undergo fermentation if infected with Z. Bailii. A concentration of 10 mmol /L is required to prevent the growth of Z. bailii. Warth (1991) investigated the benzoate resistance of Z. bailii, and reported the following results:

From data reported by Warth AD (1991) Applied and Environmental Microbiology 57: 3410-4

These results show that up to about 6 mmol/L, the cells are able to control the accumulation of benzoate, and maintain a ratio of intracellular : extracellular benzoate of about 7:1. The fall in [ATP] suggests that this may be as a result of active pumping out of the benzoate that has entered the cells.

Above about 6 mmol/L, the cells seem unable to cope with the influx of benzoate, and the ratio of intracellular : extracellular benzoate rises to 13.5 at 8 mmol /L, and as high as 17.5 at 12 mmol /L. As this occurs, there is a severe fall in the intracellular [ATP], to below the level at which the cells are able to survive.

Although there is some fall in intracellular ATP at 2 - 4 mmol/L benzoate, there is also a considerable increase in the rate of ethanol formation, reflecting increased glucose metabolism. This again suggests that the metabolism of glucose to ethanol is linked to the formation of ATP from ADP and inorganic phosphate.

Above 4 mmol/L benzoate there is a decrease in the rate of glucose metabolism, and at 8 mmol /L benzoate, when ATP falls sharply, there is a considerable reduction in glucose metabolism. This suggests that, as well as the metabolism of glucose being linked to the formation of ATP from ADP and phosphate, there is also a requirement for ATP for glucose metabolism, as seen in the studies with erythrocytes.

Haemolytic anaemia as a result of low plasma phosphate (hypophosphataemia)

During his studies, Abdul came across three case reports of haemolytic anaemia associated with low plasma concentrations of phosphate.

Lichtman et al (1969) reported on a patient with kidney failure who developed haemolytic anaemia as a result of vigorous efforts to reduce his plasma phosphate (which is commonly elevated in kidney failure) by administration of aluminium hydroxide.

From data reported by Lichtman MA et al (1969) New England Journal of Medicine 280: 240-4

Discontinuation of aluminium hydroxide led to increase in plasma phosphate and erythrocyte ATP to within normal range over 8 days.

Jacob & Amsden (1971) reported on a haemolytic crisis associated with hypophosphataemia occurring in association with heavy drinking over a period of several years. Over the 21 days after hospitalization and with intravenous feeding, there was a gradual repletion of serum phosphate. They reported the following findings:

From data reported by Jacob HS & Amsden T (1971) New England Journal of Medicine 285: 1446-50

Severe hypophosphataemia leads to a fall in red cell ATP, suggesting that there is a requirement for inorganic phosphate for the synthesis of ATP.

This is confirmed by the recovery of normal erythrocyte ATP in parallel with the recovery in plasma phosphate in the second patient.

ATP seems to be required for maintenance of normal erythrocyte integrity.

Lichtman et al (1971) reported an acute haemolytic crisis in a patient with severe intestinal disease who was given intravenous glucose and amino acids at a high level. A baseline blood sample was taken on admission to the intensive care unit, a second sample after 72 hours of intravenous nutrition, and a third after 24 hours of intravenous nutrition with an infusion of inorganic phosphate.

From data reported by Lichtman MA et al (1971) Annals of Internal Medicine 74: 562-8

This again confirms the importance of inorganic phosphate for the maintenance of erythrocyte ATP, which falls on hypophosphataemia associated with the (phosphate-free) intravenous nutrition, and rises again when phosphate is added to the intravenous fluid.

The reason for the fall in ATP seems to be a marked reduction in the rate of glucose utilisation and lactate formation, suggesting that there is a requirement for either ATP or inorganic phosphate (or both) for the metabolism of glucose.

The need for phosphate for glucose metabolism

Tsuboi and Fukunaga (1965) studied the effect of varying the concentration of phosphate on glucose metabolism in erythrocytes.

From data reported by Tsuboi KK & Fukunaga K (1965) Journal of Biological Chemistry 240: 2806-10

Phosphate stimulates utilisation of glucose and formation of lactate, but, while at low concentrations of phosphate there is the expected ratio of 2 mol of lactate formed per mol of glucose consumed, at high concentrations of inorganic phosphate, less lactate is formed than would be expected.

The ratio of lactate formed : glucose consumed falls from 2.0 at up to 2 mmol /L phosphate, to 1.6 at 10 mmol /L phosphate, and 1.3 at 50 mmol /L This suggests either:

• Phosphate stimulates some other pathway for the disappearance of lactate (this could be checked by incubating with lactate)
• There is increased accumulation of metabolic intermediates in the presence of additional phosphate.

In a further experiment they measured the change in concentration of inorganic phosphate and acid-soluble organic phosphates, glucose utilization, and lactate formation in erythrocytes incubated with and without added phosphate in the medium.

From data reported by Tsuboi KK & Fukunaga K (1965) Journal of Biological Chemistry 240: 2806-10

This clearly shows that in the presence of additional phosphate there is an accumulation of organic phosphate (which must be assumed to be phosphorylated intermediates of the pathway). This presumably accounts for the lower than expected ratio of lactate formed / glucose consumed.

Note that in the absence of added phosphate, there was more lactate formed than can be accounted for by the glucose consumed (lactate / glucose ratio = 2.35). This was accompanied by a loss of organic phosphate, suggesting that the additional lactate has been formed from phosphorylated intermediates normally present in the cells.

Key points from this exercise:

• It is normal that lactase activity in the small intestine is lost during late adolescence; among people of northern European origin (and some other groups) the enzyme persists into adult life.
• People who lack lactase are intolerant of lactose, and a moderately large amount causes bloating, flatulence and diarrhoea because of the metabolism of unabsorbed lactose by intestinal bacteria.
• Under anaerobic conditions, yeast ferments glucose to yield 2 mol of ethanol and 2 mol of carbon dioxide per mol of glucose. The initial series of reactions leads to formation of 2 mol of a 3-carbon compound (pyruvate), which then undergoes decarboxylation and reduction to ethanol and carbon dioxide.
• Fluoride inhibits the metabolism of glucose in bacteria and red blood cells. Blood samples for measurement of glucose are collected in tubes containing sodium fluoride.
• Red blood cells metabolise glucose to form 2 mol of lactate per mol of glucose. The amount of glucose metabolised depends on the amount of ADP provided.
• Two mol of ATP are formed for each mol of glucose metabolised to lactate. However, in red cells that have been depleted of ATP there is no metabolism of glucose - ATP is also required for glucose metabolism.
• Glucose is taken up by red blood cells by metabolic trapping - it crosses the cell membrane by facilitated diffusion and is then phosphorylated to glucose 6-phosphate, which cannot cross the cell membrane, at the expense of ATP.
• Inorganic phosphate is also required for glucose metabolism and ATP formation in red blood cells. At low concentrations of phosphate glucose metabolism is impaired. At high concentrations of phosphate a number of phosphorylated intermediates accumulate. You will investigate these intermediates and deduce the pathway of glucose metabolism in later exercises.