Laboratory Diagnosis in Neurology, 1 Ed.

Special Serum Analysis

M. Uhr

Determination of Drug Levels

Therapeutic drug monitoring (TDM). Therapeutic drug monitoring usually relates to the determination of medicinal drug levels in blood and plasma and is intended to help the physician to optimize pharmacotherapy, i. e., to avoid ineffective treatment as much as to avoid side effects. It must be remembered that patients with similar diseases and similar symptoms often have qualitatively and quantitatively different reactions to the same drug. Without TDM, patients who do not respond to medical therapy because they break down the drug too rapidly, or patients who develop very high blood levels after minimal drug doses because of their own individual disposition and interactions, would remain unrecognized. TDM thus helps to avoid relapses (e. g., epileptic seizures, depression) and to save treatment costs.

In TDM, the concentrations of the active substance and its pharmacologically effective metabolites in the blood are determined, and the patient's dosage is then adjusted until effective concentrations are reached at which a therapeutic response may be expected but side effects should not occur. TDM thus demonstrates the pharmacogenetic profile of the individual patient.

Indication. In neurology and psychiatry, drug levels are determined for anticonvulsants, antidepressants, neuroleptics, and phase prophylactics, including lithium (Table 6.1). In particular, TDM is used whenever the success of treatment cannot be directly evaluated on the basis of the patient's clinical symptoms. This is the case for anticonvulsants in patients with a relatively low seizure frequency, and also for antidepressants, which take days or weeks to start working. TDM is indicated when:

• Side effects occur even at usual clinical dose.

• The patient is responding inadequately to the drug.

• Seizure frequency increases on anticonvulsants.

• Noncompliance is suspected.

Table 6.1 Case-related indications for psychopharmaceutical TDM

• Noncompliance

• No response or inadequate response to the usual clinical dose

• Pronounced side effects with the usual clinical dose

• Combination treatment with a drug with a known potential for pharmacokinetic interaction

• Relapse while on maintenance regimen

• Known pharmacogenetic factors

• Children and adolescents

• Patients over 60

• Forensic indications

Preanalytical requirements. At the time of blood collection, base levels of the drug under steady-state conditions should have been established. About 5–10 mL serum or plasma is usually required.

Analysis. Various analytical methods are available for determining drug levels; among them are immunological methods in which the drug molecules act as antigens and are demonstrated using the appropriate antibody and subsequent detection by UV light absorption or fluorescence. Anticonvulsants like carbamazepine and valproic acid are determined in automated immunological assays. One problem with psychopharmaceutical TDM is that blood concentrations of the drugs are often very low. In such cases, chromatographic methods are used:

• For chromatographic separation: liquid chromatography (HPLC) or gas chromatography (GC).

• For substance-dependent detection: UV light absorption, fluorescence, electrochemical detection, or mass spectrometry (MS).

A problem with immunological methods is cross-reactivity of the immunoassays. Among chromatographic procedures, mass spectrometry has become more prominent in recent years: in particular, individual substances can be very sensitively and specifically detected by liquid chromatography –mass spectrometry (LC/MS/MS technique). Sometimes detection of enantiomers is required. For this purpose, stereoselective derivatization or separation by chiral chromatography (GC or HPLC) is used.

Interpretation. TDM involves not only the determination of drug levels in the blood but also their interpretation in the wider context. For this purpose, a properly completed request form is needed that states clearly what information is required and why, and supplies details of medication and supporting medication. Drug levels must be related to therapeutic ranges, although these therapeutic ranges should not be taken as absolutes but—depending on the drug—as guide values. The main issue for therapeutic decision-making is the overall clinical picture, i. e., whether the patient is already responding to the drug and/or whether side effects are already occurring. Therapeutic ranges for anticonvulsants are given in Table 6.2, and those for antidepressants/neuroleptics are given in Table 6.3.

Table 6.2 Therapeutic ranges for anticonvulsants

Substance

Serum concentration, therapeutic range, μg/mL

Carbamazepine (total concentration)

4–10

Carbamazepine (free fraction)

<1.5 (1.5 is the upper limit before side effects appear)

Ethosuximide

40–100

Felbamate

20–80

Gabapentin

2–21

Lamotrigine

3–15 (not precisely known; levels up to 17 μg/mL are tolerated without side effects)

Phenobarbital

10–40

Phenytoin (total concentration)

10–20

Phenytoin (free fraction)

1.2–2.5

Primidone

5–15

Tiagabine

Unknown

Topiramate

Unknown

Valproic acid

50–100

Table 6.3 Therapeutic ranges for psychopharmaceuticals (Hiemke and Härtter, 2000; modified in accordance with the AGNP-TDM expert group consensus guidelines on therapeutic drug monitoring, Baumann et al., 2004)

Substance

Serum concentration at effective doses (ng/mL, unless indicated otherwise)

Antidepressants

Amitriptyline

100–220

Citalopram

30–130

Clomipramine

175–450

Desipramine

100–300

Doxepin

50–150

Escitalopram

15–80

Fluoxetine

120–300

Fluvoxamine

150–300

Imipramine

175–300

Maprotiline

125–200

Mianserin

15–70

Mirtazapine

40–80

Moclobemide

300–1000

Nefazodone

100–600

Nortriptyline

70–170

Paroxetine

70–120

Reboxetine

10–100

Sertraline

10–50

Trazodone

650–1500

Trimipramine

150–350

Venlafaxine

200–400

Viloxazine

20–500

Antipsychotics

Amisulpride

100–400

Benperidol

2–10

Chlorpromazine

30–300

Clozapine

350–600

Fluphenazine

0.5–2

Flupentixol

> 2

Haloperidol

5–17

Levomepromazine

15–60

Olanzapine

20–80

Perazine

100–230

Perphenazine

0.6–2.4

Quetiapine

70–170

Risperidone

20–60

Sulpiride

200–1000

Thioridazine

200–2000

Zotepine

12–120

Phase prophylactics

Carbamazepine

6–12 μg/mL

Valproic acid

50–100 μg/mL

Lithium

0.5–1.2 mmol/L

Antidementia drugs

Donepezil

30–75

Galantamine

30–100

Memantine

7–159

Tacrine

7–30

References

Baumann P, Hiemke C, Ulrich S, et al. The AGNP-TDM expert group consensus guidelines: therapeutic drug monitoring in psychiatry. Pharmacopsychiatry 2004;37:243–265.

Hiemke C, Härtter S. Pharmacokinetics of selective serotonin reuptake inhibitors. Pharmacol Ther 2000;85:11–28.

Intoxication

In every patient presenting with sudden health impairment, the differential diagnosis should include acute intoxication. Cases of exogenous intoxication constitute 5–10% of all admissions to medical clinics. They are the most common cause of nontraumatic coma in adults.

Etiology. Causes of intoxication vary. In psychiatric patients, they are often suicide attempts, with ingestion of overdoses of hypnotics and tranquilizers being the most common cause.

Clinical picture. Common symptoms of intoxication include reduced consciousness or coma, changes in pupil reactivity and contraction, vomiting, diarrhea, nausea, hyperventilation, psychosis, ataxia, myoclonia, and epileptic seizures. Paralysis, respiratory and cardiovascular disturbances, and gastrointestinal and dermatological symptoms may occur.

Diagnosis. If there is no clue as to the type of the intoxication, a systematic toxicological laboratory diagnosis should be carried out, rather than a specific analysis of the noxa. For this purpose, serum, urine, and stomach content are analyzed. If some clue regarding the type of intoxication, changes in the concentration of the substances in question in the blood and urine should be followed using appropriate methods. In addition, laboratory diagnosis of intoxication includes blood count, blood sugar, electrolytes, blood clotting (Quick test and PTT), creatinine, liver function, creatine kinase, lactate, ammoniac, alcohol level, blood gas analysis (to identify metabolic acidosis), and urinalysis. Comprehensive toxicological screening is recommended. Rapid tests are available for this, which identify more than 90% of intoxicating substances. Most of these tests are commercially available assays based on immune reactions. Rapid tests are available for opiates, benzodiazepines, barbiturates, tricyclic antidepressants, cocaine, amphetamines, cannabinoids, and other substances. For many drugs (see above, “Determination of Drug Levels”) and toxic substances, serum levels decide the indication for and use of antidote treatment.

Therapy. Treatment is normally initiated before toxicology results are available. Poison control centers provide information about procedures in cases of intoxication.

Determination of Vitamins

Vitamins are essential nutrients because the human metabolism does not synthesize them. They catalyze numerous biochemical reactions in cells. An adequate daily supply of vitamins is required for vitamin-mediated enzyme reactions without complications.

Hypovitaminosis. Insufficient supply and depletion of endogenous depots cause characteristic deficiency diseases, collectively called hypovitaminosis. Severe hypovitaminosis affects the whole body; this chapter describes only neuropsychiatric disorders. In Western Europe and North America, hypovitaminosis is rare. It is mainly seen in patients who abuse alcohol, recreational drugs, or medication, or as a result of poor nutrition during dieting, malnutrition in old age, or increased vitamin requirement during pregnancy and lactation. Neurological or psychiatric symptoms arise from deficiencies in vitamins A, B1, B6, B12, D, and E, biotin, nicotinic acid, and pantothenic acid.

Hypervitaminosis. Hypervitaminosis is known for vitamins A, B6, and D.

Vitamin A (Retinol)

Etiology. The causes of this hypovitaminosis include poor intestinal absorption, impaired conversion of carotene (the provitamin) to retinol, reduced availability because of liver disease, and increased requirement.

Pathophysiology. When plasma levels of retinol drop below 100–200 μg/L, hypovitaminosis develops. The plasma level starts to drop only after extensive depletion of the liver, which has a reserve capacity of 1–2 years.

Clinical picture. Hypovitaminosis leads to changes in the retina, skin, and mucosae. Early symptoms are impaired light and dark adaptation leading to night blindness (nyctalopia), which is accompanied by increased sensitivity to glare. Abnormal keratinization in the form of softening of the cornea (keratomalacia) and dryness of cornea and conjunctiva (xerophthalmia) leading to loss of sight may also occur. Optic nerve atrophy and impaired CSF absorption with symptoms of cerebral pressure and epileptic seizures have been reported.

Analysis. Methods for determining retinol and carotene usually include HPLC with detection of UV light absorption or, better, fluorescence (Table 6.4).

Vitamin B1 (Thiamine)

Etiology. Like folic acid deficiency, thiamine deficiency is relatively frequent in all age groups. The main causes are inadequate nutrition or malnutrition when on a diet or in old age, chronic alcohol abuse, increased requirement during pregnancy and lactation, and malabsorption syndromes. Congenital disorders of thiamine metabolism are also known.

Table 6.4 Hypovitaminosis

Vitamin

Hypovitaminosis symptoms

Laboratory diagnosis

Vitamin A (retinol)

Night blindness, softening of the cornea (keratomalacia), optic nerve atrophy, impaired CSF absorption, increased intracranial pressure

• Serum retinol ↓

– Method: HPLC

– Reference range: 200–600 μg/L

Vitamin B1 (thiamine)

Wernicke's encephalopathy, Korsakov's syndrome, polyneuropathy (PNP), poor concentration, depression

• Whole blood and serum thiamine ↓

– Method: HPLC

– Reference range:

EDTA blood: 19–49 μg/L

Serum: 1.3–7.5 μg/L

Vitamin B6 (pyridoxine)

PNP, epilepsy, depression, disorientation

• Plasma pyridoxal phosphate ↓

– Method: HPLC

– Reference range: 1–2.4 μg/L (39–98 nmol/L)

• Tryptophan load test

Vitamin B12 (cobalamin)

Encephalopathy, psychosis, funicular myelosis, sensorimotor PNP

• Serum and plasma vitamin B12 ↓

– Method: immunological assays

– Reference range: 0.2–1 μg/L (148–738 pmol/L)

• Homocysteine and methylmalonic acid ↑

– Method: GC and MS

– Reference range: methylmalonic acid, 53–376 nmol/L; total cysteine, 4.1–21.3 μmol/L

Folate

Funicular myelosis, cognitive dysfunction, neuropathy, depression, low folic acid levels are often associated with vitamin B12 deficiency

• Serum folate ↓

– Method: HPLC: immunological assays with various means of detection

– Reference range: 2–9.1 μg/L (3–30 nmol/L)

Pantothenic acid

Paresthesia, burning feet syndrome, cramps, insomnia, exhaustion

• Serum pantothenic acid ↓

– Method: RIA, HPLC

– Reference range: 200–1100 μg/L

Niacin

Cutaneous and mucosal changes, encephalitis, extrapyramidal symptoms, myelopathies

Urinary excretion of niacin metabolites ↓

Vitamin C (ascorbic acid)

Scurvy, hemorrhage, behavioral changes

• Serum vitamin C ↓

– Method: HPLC

– Reference range: 4–18 mg/L (20–100 μmol)

Vitamin D (calciferol)

Rickets, myopathy, tetany

• Serum 25-hydroxycholecalciferol ↓

– Method: immunoassays

– Reference range: 10–44 μg/L

Vitamin E (α-tocopherol)

Spinocerebellar degeneration, retinitis pigmentosa

• Serum tocopherol ↓

– Method: HPLC

– Reference range: 5–20 mg/L

Clinical picture. Early symptoms of thiamine deficiency include psychological disturbances with irritability, lack of concentration, memory problems, and depression. Mild chronic deficiency mainly leads to beriberi polyneuropathy, whereas pronounced hypovitaminosis causes heart failure and Wernicke's encephalopathy.

Analysis. Vitamin B1 in whole blood, serum, and urine is determined by chromatography (HPLC). It is assumed that there is a correlation between the levels in blood and tissue, although only 0.8% of the total body thiamine is present in the blood. There is a marked difference between whole blood and serum (Table 6.4), since most of the thiamine is in cells.

A 30–50% reduction in erythrocyte transketolase also indicates thiamine deficiency.

Vitamin B6 (Pyridoxine)

Vitamin B6 is the term used to refer collectively to pyridoxine and its derivatives. Pyridoxal phosphate is considered the most important active form of the vitamin.

Etiology. Isolated vitamin B6 deficiency is rare; it is usually the result of inadequate supply of vitamin B complex. In addition to insufficient supply and poor intestinal absorption, particularly in alcoholics, a deficiency syndrome can be the result of ingestion of pyridoxine antagonists. These include anticonvulsants, isoniazid, penicillamine, estrogencontaining contraceptives, tranquilizers, and various headache remedies.

Clinical picture. Vitamin B6 deficiency affects mostly the central nervous system and the peripheral nerves. It causes nervousness, depression, disorientation, and, later, epileptic seizures. Distal sensorimotor polyneuropathy may develop. Seborrheic dermatitis and hypochromic microcytic anemia have also been reported.

A very high supply of this vitamin may cause hypervitaminosis with polyneuropathy and ataxia.

Analysis. Vitamin B6 metabolism can be assessed by determining the level of pyridoxal phosphate in the plasma and of pyridoxic acid in the urine (> 3 μmol/d for the latter). HPLC methods are used for this purpose. For an indirect functional test, activity of aspartate aminotransferase in erythrocyte lysate with and without the addition of pyridoxal phosphate is determined. The tryptophan load test, in which the excretion of xanthurenic acid in the urine is measured after administration of a defined oral dose of tryptophan, is rarely used.

Vitamin B12 (Cobalamin)

Etiology. Vitamin B12 deficiency is caused by alcoholism, achlorhydria, intrinsic factor deficiency, antibodies against vitamin B12, and impaired absorption.

Pathophysiology. Vitamin B12-dependent enzymes include methylmalonyl-CoA mutase and homocysteine methyltransferase. In cobalamin deficiency, the metabolic steps catalyzed by these enzymes are inhibited, and the substrates methylmalonic acid (MMA) and homocysteine (HC) accumulate in the blood. Diagnosis of vitamin B12 deficiency is thus possible by measuring the vitamin concentration as well as the concentrations of MMA and HC in the serum or plasma.

Clinical picture. Vitamin B12 deficiency manifests itself in neurological, hematological, and gastrointestinal symptoms. Disturbances of the central and peripheral nervous systems include encephalopathy, myelopathy, and sensorimotor polyneuropathy. Psychopathological disorders, cognitive dysfunction, and even productive organic psychosis may occur. In funicular myelosis, foci of demyelination of the posterior fascicles and pyramidal tracts develop, and the resulting symptoms dominate the picture.

Analysis. Vitamin B12 is mostly determined by immunological tests, such as radioimmunoassays or chemiluminescence immunoassays. Methylmalonic acid and homocysteine are measured by gas chromatography, mass spectrometry, or HPLC (Table 6.4).

Interpretation. Deficiency is ruled out at vitamin B12 concentrations above 220 pmol/L. Treatment should be undertaken when the concentration is between 150 and 220 pmol/L and MMA and HC levels in the blood are elevated. Vitamin B12 levels below 150 pmol/L indicate deficiency. If deficiency is detected, vitamin B12 absorption tests (Schilling test) are carried out; more than 10% of the 57Colabeled vitamin B12 administered should be excreted within 24 hours.

Folate

Folates are involved in many different biochemical reactions. Folate deficiency is one of the most common vitamin deficiencies worldwide and is found especially in pregnant women, adolescents, and elderly persons.

Etiology. Causes of folate deficiency include insufficient supply, impaired absorption, increased requirement during pregnancy, and ingestion of folate antagonists (e. g., anticonvulsants, methotrexate).

Pathophysiology. About 50% of the folate in the body is stored in the liver. Deficiency develops only 3–6 months after the supply has stopped.

Clinical picture. Folate deficiency leads to impairment of the hematopoietic system and to gastrointestinal and neuropsychiatric symptoms. These include dementia, depression, and sensory polyneuropathy.

Analysis. The available methods for determining folate levels include ligand assays and various immunological tests. A confounding factor in the analysis may be hemolysis, which releases folates from erythrocytes, leading to falsely high serum values. Serum samples should be protected from light. Reduced folate concentrations can also be a result of vitamin B12 deficiency (Table 6.4). It should be noted that the results obtained by different commercial folate assays vary considerably.

Vitamin E (Tocopherol)

Etiology. Possible causes of vitamin E deficiency include reduced absorption, genetic defects of α-tocopherol transport protein, or abetalipoproteinemia.

Clinical picture. Vitamin E hypovitaminosis causes spinocerebellar degeneration with cerebellar ataxia, dysarthria, and impaired proprioception and vibration perception. Proximal paresis and pyramidal tract symptoms also occur.

Analysis. Vitamin E is mostly determined by HPLC and fluorescence measurement. The important value is the ratio of vitamin E concentration to lipid concentration in the serum (> 0.8 is normal). Serum vitamin E levels are in the range of 5–20 mg/L (Tables 6.4 and 21.9).