Chapter 57: Biochemical Case Histories

In this final chapter, nine case histories are presented as open-ended problems for you to solve, based on what you have learnt from studying this book. No solutions are provided, and there is no discussion of the cases; all that you need to know in order to explain the problems is available elsewhere in this book.

In many cases the patient’s clinical chemistry results are presented together with reference ranges. These may differ from problem to problem, and from the reference ranges in Table 48–3, because, as discussed in Chapter 48, reference ranges from different laboratories differ in many cases.

The patient is a 5-year-old boy, who was born in 1967, at term, after an uneventful pregnancy. He was a sickly infant, and did not grow well. On a number of occasions his mother noted that he appeared drowsy, or even comatose, and said that there was a “chemical, alcohol-like” smell on his breath, and in his urine. The GP suspected diabetes mellitus, and sent him to the Middlesex Hospital in London for a glucose tolerance test. The results are shown in Figure 57–1.

Blood samples were also taken for measurement of insulin at zero time and 1 hour after the glucose load. At this time a new method of measuring insulin was being developed, radioimmunoassay (see Chapter 48), and therefore both this and the conventional biological assay were used. The biological method of measuring insulin is by its ability to stimulate the uptake and metabolism of glucose in rat muscle in vitro; this can be performed relatively simply by measuring the radioactivity in ¹⁴CO₂ after incubating duplicate samples of the muscle with [¹⁴C]glucose, with and without the sample containing insulin. The results are shown in Table 57–1.

As a part of their studies of the new radioimmunoassay for insulin, the team at the Middlesex Hospital performed gel exclusion chromatography of a pooled sample of normal serum, and determined insulin in the fractions eluted from the columns both by radioimmunoassay (graph A in Figure 57–2) and by stimulation of glucose oxidation (graph B). Three molecular mass markers were used; they eluted as follows: M_r 9000 in fraction 10, M_r 6000 in fraction 23, and M_r 4500 in fraction 27.

The investigators also measured insulin in the fractions eluted from the chromatography column after treatment of each fraction with trypsin. The results are shown in graph C.

After seeing the results of these studies, they subjected the same-pooled serum sample to brief treatment with trypsin, and performed gel exclusion chromatography on the product. Again they measured insulin by radioimmunoassay (graph D) and biological assay (graph E).

Since these studies in the 1960s, the gene for human insulin has been cloned. Although insulin consists of two peptide chains, 21 and 30 amino acids long, respectively, these are coded for by a single gene, which has a total of 330 base pairs between the initiator and stop codons. As you would expect for a secreted protein, there is a signal sequence coding for 24 amino acids at the 5′ end of the gene.

The patient is a 50-year-old man, 174 cm tall and weighing 105 kg. He is an engineer, and works on secondment in one of the strict Islamic states in the Gulf, where alcohol is prohibited. At the beginning of August he returned home for his annual leave. According to his family, he behaved as he usually did when on leave, consuming a great deal of alcohol and refusing meals. He was known to be drinking 2 L of whiskey, two or three bottles of wine, and a dozen or more cans of beer each day; his only solid food consisted of sweets and biscuits.

On September 1st he was admitted to the emergency department, semiconscious, and with a rapid respiration rate (40/minute). His blood pressure was 90/60 and his pulse rate was 136/minute. His temperature was normal (37.1°C). Emergency blood gas analysis revealed severe acidosis: pH 7.02 and base excess −23; pO₂ 91 mm Hg and pCO₂ 10 mm Hg. He was transferred to intensive care and given intravenous bicarbonate.

His pulse rate remained high, and his blood pressure low, so emergency cardiac catheterization was performed; this revealed a cardiac output of 23 L/min (normal 4-6). A chest x-ray shows significant cardiac enlargement.

Table 57–2 shows the clinical chemistry results from a plasma sample taken shortly after he was admitted.

The patient is an African American recruit to the army. He was given the antimalarial drug primaquine, and suffered a delayed reaction with kidney pain, dark urine, and low red blood cell counts that led to anemia and weakness. Centrifugation of a blood sample showed a low hematocrit, and the plasma was red colored.

Similar acute hemolytic attacks have been observed, predominantly in men of Afro-Caribbean origin, in response to primaquine and a variety of other drugs, including dapsone, the antipyretic acetylphenylhydrazine, the antibacterial bactrim/septrin, sulfonamides, and sulfones, whose only common feature is that they all undergo cyclic nonenzymic reactions in the presence of oxygen to produce hydrogen peroxide and a variety of oxygen radicals that can cause oxidative damage to membrane lipids, leading to hemolysis. Moderately severe infection can also precipitate a hemolytic crisis in susceptible people.

One way of screening for sensitivity to primaquine is based on the observation that the glutathione concentration of erythrocytes from sensitive subjects is somewhat lower than that of control subjects, and falls considerably on incubation with acetylphenylhydrazine.

Glutathione (GSH) is a tripeptide, γ-glutamyl-cysteinyl-glycine, which readily undergoes oxidation to form a disulphide-linked hexapeptide, oxidized glutathione, generally abbreviated to GSSG. Table 57–3 shows the concentrations of GSH and GSSG in red cells from the patient and 10 control subjects, before and after incubation with acetylphenylhydrazine.

The reported K_m of glutathione reductase for GSSG is 65 μmol/L and for NADPH 8.5 μmol/L. Erythrocyte lysates were incubated with a saturating concentration of GSSG (1 mmol/L) and either NADPH or NADH (100 μmol/L). Each incubation contained the hemolysate from 0.5-mL packed cells (Table 57–4).

Since none of the red cell lysates showed any significant activity with NADH, it is unlikely that there is any transhydrogenase activity in erythrocytes, to reduce NADP⁺ to NADPH at the expense of NADH. This raises the problem of the source of NADPH in erythrocytes.

The dye methylene blue will oxidize NADPH; the reduced dye then undergoes nonenzymic oxidation in air, so the addition of a relatively small amount of methylene blue will effectively deplete NADPH, and would be expected to stimulate any pathway that reduces NADP⁺ to NADPH.

Erythrocytes from control subjects were incubated with 10 mmol/L [¹⁴C]glucose with or without the addition of methylene blue; all six possible positional isomers of [¹⁴C]glucose were used, and the radioactivity in (lactate + pyruvate) was determined after thin layer chromatography of the incubation mixture. Each incubation contained 1 mL erythrocytes in a total incubation volume of 2 mL (Table 57–5).

In further studies, only the formation of ¹⁴CO₂ from [¹⁴C-1]glucose was measured, with the addition of:

• Sodium ascorbate (which undergoes a nonenzymic reaction in air to produce H₂O₂)

• Acetylphenylhydrazine (which is known to precipitate hemolysis in sensitive subjects, and depletes reduced glutathione)

• Methylene blue (which oxidizes NADPH)

The incubations were repeated with N-ethylmaleimide, which undergoes a nonenzymic reaction with the —SH group of reduced glutathione, and thus depletes the cell of total glutathione. The results are shown in Table 57–6.

Further studies showed that the patient’s red blood cells contained only about 20% of the normal activity of glucose 6-phosphate dehydrogenase (see Chapter 20). In order to investigate why his enzyme activity was so low, a sample of his red blood cells was incubated at 45°C for 60 minutes, then cooled to 30°C and the activity of glucose 6-phosphate dehydrogenase was determined. After the preincubation at 45°C, his red cells showed only 60% of their initial activity. By contrast, red cells from control subjects retained 90% of their initial activity after preincubation at 45°C for 60 minutes.

The patient is a 10-year-old Maltese boy. On his birthday his aunt gave him a pie made from fava beans (a local delicacy), and that evening he suffered kidney pain, and passed dark urine. A blood film showed a low red blood cell count and the plasma was red colored. This problem is not uncommon in Malta, and indeed several of his classmates (all boys) have died when an acute crisis has been precipitated by eating fava beans, or after a moderate fever associated with an infection.

Further studies showed that his erythrocyte glucose 6-phosphate dehydrogenase was only 10% of normal and had a very high K_m for NADP⁺. Unlike the patient in case 3, his red blood cell enzyme was as stable to incubation at 45°C as that from control subjects.

The patient is a 28-week-old baby girl. She was admitted to the emergency department in a coma, having suffered a convulsion after feeding. She had a mild infection and slight fever at the time. Since birth she had been a sickly child, and had frequently vomited and become drowsy after feeding. She was bottle-fed and at one time cows’ milk allergy was suspected, although the problems persisted when she was fed on soya-milk.

On admission she was mildly hypoglycemic, ketotic and her plasma pH was 7.29. Analysis of a blood sample showed normal levels of insulin, but considerable hyperammonemia (plasma ammonium ion concentration 500 μmol/L; reference range 40-80 μmol/L). She responded well to intravenous glucose infusion and rectal infusion of lactulose, regaining consciousness. She had poor muscle tone.

A liver biopsy sample was taken, and the activities of the enzymes of urea synthesis (see Chapter 28) were determined, and compared with activities in postmortem liver samples from six infants of the same age. The results are shown in Table 57–7. She remained well on a high carbohydrate, low protein diet for several days, although the poor muscle tone and muscle weakness persisted. A second liver biopsy sample was taken after 4 days and the activity of the enzymes determined again.

Her very low protein diet was continued, but in order to ensure an adequate supply of essential amino acids for growth she was fed a mixture of the keto-acids of threonine, methionine, leucine, isoleucine, and valine. After each feed she again became abnormally drowsy and markedly ketotic, with significant acidosis. Her plasma ammonium ion concentration was within the normal range, and a glucose tolerance test was normal, with a normal increase in insulin secretion after glucose load.

High-pressure liquid chromatography of her plasma revealed an abnormally high concentration of propionic acid (24 μmol/L; reference range 0.7-3.0 μmol /L). Urine analysis showed considerable excretion of methylcitrate (1.1 μmol/mg creatinine), which is not normally detectable. She was also excreting a significant amount of short-chain acyl carnitine (mainly propionyl carnitine)—28.6 μmol/24 hours, compared with a reference range of 5.7 + 3.5 μmol/24 hours.

The metabolism of a test dose of [¹³C]propionate given by intravenous infusion was determined in the patient, her parents and a group of control subjects; skin fibroblasts were cultured and the activity of propionyl CoA carboxylase was determined by incubation with propionate and NaH¹⁴CO₃, followed by acidification and measurement of the radioactivity in products. The results are shown in Table 57–8.

The results of measuring carnitine in the first liver biopsy sample and in a muscle biopsy sample gave the results shown in Table 57–9.

The patient is a 9-month-old girl, the second child of unrelated parents. She was born at term after an uneventful pregnancy, weighing 3.4 kg and was breast fed, with gradual introduction of solids from 3 months of age onward. Her mother reported that, while she liked cheese, meat, and fish, she frequently became irritable and grizzly after meals, and became lethargic, drowsy, and “floppy” after eating relatively large amounts of protein-rich foods. Her urine had a curious odor, described by her mother as being “cat-like,” on such occasions.

At 9 months of age she was admitted to the emergency department in a coma, and suffering convulsions. She had been unwell for the last 3 days, with a slight fever, and for the last 12 hours had been refusing all food and drink. At this time she weighed 8.8 kg, and her body length was 70.5 cm.

Emergency blood tests revealed moderate acidosis (pH 7.25) and severe hypoglycemia (glucose <1 mmol/L); a dipstick test for plasma ketone bodies was negative. A blood sample was taken for full clinical chemistry tests, and she was given intravenous glucose. Within a short time she recovered consciousness. The results of the blood tests are shown in Table 57–10.

She remained in hospital for several weeks, while further tests were performed. She was generally well through this time, but became drowsy and severely hypoglycemic, and hyperventilated, if she was deprived of food for more than about 8 to 9 hours. Her muscle tone was poor, and she was very weak, with considerably less strength (eg, in pushing her arms or legs against the pediatrician’s hand) than would be expected for a girl of her age.

On one occasion her blood glucose was monitored at 30-minute intervals over 3 hours from waking, without being fed. It fell from 3.4 mmol/L on waking to 1.3 mmol/L 3 hours later. She was deprived of breakfast again the next day, and again blood glucose was measured at 30-minute intervals for 3 hours during which she received an intravenous infusion of β-hydroxybutyrate (50 μmol/min/kg body weight). During the infusion of β-hydroxybutyrate her plasma glucose remained between 3.3 and 3.5 mmol/L.

At no time were ketone bodies detected in her urine, and there was no evidence of any abnormal excretion of amino acids. However, a number of abnormal organic acids were detected in her urine, including relatively large amounts of 3-hydroxy-3-methylglutaric and 3-hydroxy-3-methylglutaconic acids. The excretion of these two acids increased considerably under two conditions:

1. When she was fed a relatively high protein meal (she became grizzly, lethargic, and irritable). A blood sample taken after such a meal showed significant hyperammonemia (130 μmol/L), but normal glucose (5.5 mmol/L).

2. When she was fasted for more than the normal overnight fast, with or without the infusion of β-hydroxybutyrate.

One obvious metabolic precursor of 3-hydroxy-3-methylglutaric acid is 3-hydroxy-3-methylglutaryl CoA (HMG-CoA), which is normally cleaved to yield acetoacetate and acetyl-CoA by the enzyme hydroxymethyglutaryl CoA lyase (Chapter 22). Therefore the activity of this enzyme was determined in leukocytes from blood samples from the patient and her parents. The results are shown in Table 57–11.

Analysis of her urine also revealed considerable excretion of carnitine, as shown in Table 57–12.

The patient is a 9-month-old boy, the second child of unrelated parents; his brother is 5 years old, fit and healthy. He was born at full term after an uneventful pregnancy, weighing 3.4 kg (the 50th centile), and developed normally until he was 6 months old, after when he showed some retardation of development. He also developed a fine scaly skin rash about this time, and his hair, which had been normal, became thin and sparse.

At 9 months of age he was admitted to the emergency department in a coma; the results of clinical chemistry tests on a plasma sample are shown in Table 57–13.

The acidosis was treated by intravenous infusion of bicarbonate, and he recovered consciousness. Over the next few days he continued to show signs of acidosis (rapid respiration), and even after a meal ketone bodies were present in his urine. His plasma lactate, pyruvate, and ketone bodies remained high; plasma glucose was in the low normal range, and his plasma insulin was normal both in the fasting state and after an oral glucose load.

Urine analysis revealed the presence of significant amounts of a number of organic acids that are not normally excreted in the urine, including:

• Lactate, pyruvate, and alanine

• Propionate, hydroxypropionate, and propionyl glycine

• Methylcitrate

• Tiglate and tiglylglycine

• 3-Methyl crotonate, 3-methylcrotonylglycine, and 3-hydroxyisovalerate

His skin rash and hair loss were reminiscent of the signs of biotin deficiency (see Chapter 44), as caused by excessive consumption of uncooked egg-white. However, his mother said that he did not eat raw or undercooked eggs at all, although he was fond of hard-boiled eggs and yeast extract (which are rich sources of biotin). His plasma biotin was 0.2 nmol/L (normal >0.8 nmol/L), and he excreted a significant amount of biotin in the form of biocytin (see Figure 44–17) and small biocytin-containing peptides, which are not normally detectable in urine.

He was treated with 5 mg of biotin per day. After 3 days the abnormal organic acids were no longer detectable in his urine, and his plasma lactate, pyruvate, and ketone bodies had returned to normal, although his excretion of biocytin and biocytin-containing peptides increased. At this stage he was discharged from hospital, with a supply of biotin tablets. After 3 weeks his skin rash began to clear, and his hair loss ceased.

Three months later, at a regular out-patient visit, it was decided to cease the biotin supplements. Within a week the abnormal organic acids were again detected in his urine, and he was treated with varying doses of biotin until the organic aciduria ceased. This was achieved at an intake of 150 μg/d (compared with the reference intake of 10-20 μg/d for an infant under 2 years old).

He has continued to take 150 μg of biotin daily, and has remained in good health for the last 4 years.

The patient is a 4-year-old girl, the only child of nonconsanguineous parents, born at term after an uneventful pregnancy. At 14 months of age she was admitted to hospital with a 1-day history of persistent vomiting, rapid shallow respiration, and dehydration. On admission, her respiration rate was 60/minute and her pulse 178/minute. The first column in Table 57–14 shows the results of clinical chemistry tests at that time. She responded rapidly to intravenous bicarbonate and a single intramuscular injection of insulin.

The results of a glucose tolerance test 3 days after admission were normal, and her plasma insulin response to an oral glucose load was within the normal range. She was discharged from hospital 7 days after admission, apparently fit and well. The second column in Table 57–14 shows the results of clinical chemistry tests taken shortly before her discharge.

She was readmitted to hospital at 16, 25, 31, and 48 months of age, suffering from restlessness, unsteady gait, rapid shallow respiration, persistent vomiting, and dehydration. Each time the crisis was preceded by a common childhood illness and decreased appetite, and she responded well to intravenous fluids and bicarbonate. A number of milder episodes were treated at home by oral fluid and bicarbonate.

During her admission at age 25 months, a skin biopsy was taken, fibroblasts were cultured, and the mitochondrial enzyme activities shown in Table 57–15 were determined.

The patient is a 5-year-old boy who is diabetic. There is a family history of diabetes, which strongly suggests a dominant pattern of inheritance. He secretes a significant amount of insulin, although less than normal subjects, suggesting that the problem is not type 1 diabetes. Unlike type 2 diabetes, this condition develops in early childhood, and is generally referred to as maturity-onset diabetes of the young (MODY).

The results of studies of the secretion of insulin by rabbit pancreas incubated in vitro with two concentrations of glucose, with and without the addition of the 7-carbon sugar mannoheptulose, which is an inhibitor of the phosphorylation of glucose to glucose-6-phosphate are shown in Table 57–16.

Two enzymes catalyze the formation of glucose 6-phosphate from glucose (see Chapter 17):

• Hexokinase is expressed in all tissues; it has a K_m for glucose of ~0.15 mmol/L

• Glucokinase is expressed only in liver and the β-cells of the pancreas; it has a K_m for glucose of ~20 mmol/L

The normal range of plasma glucose is between 3.5 and 5 mmol/L, rising in peripheral blood to 8 to 10 mmol/L after a moderately high intake of glucose. After a meal, the concentration of glucose in the portal blood, coming from the small intestine to the liver, may be considerably higher than this.

Froguel and coworkers (1993) reported studies of the glucokinase gene in a number of families affected by MODY, and also in unaffected families. They published a list of 16 variants of the glucokinase gene, shown in Table 57–17. All their patients with MODY had an abnormality of the gene.

The same authors also studied the secretion of insulin in response to glucose infusion in patients with MODY and control subjects. They were given an intravenous infusion of glucose; the rate of infusion was varied so as to maintain a constant plasma concentration of glucose of 10 mmol/L. Their plasma concentrations of glucose and insulin were measured before and after 60 minutes of glucose infusion; the results are shown in Table 57–18.