Oxford Handbook of Clinical and Laboratory Investigation

Investigations for endocrine disease have caused a lot of confusion in the minds of clinicians (many still do!). Tests have come and gone over the years and have been adopted with varying degrees of enthusiasm by specialist centres. In particular, there is often confusion over which tests to do, what procedures to follow, and how to interpret the results. In some areas (e.g. Cushing’s syndrome), controversy persists among the experts. In others, a clear consensus approach exists.

Some useful general principles

1.Use dynamic tests, rather than random (untimed) sampling where the hormone under investigation is secreted in infrequent pulses (e.g. GH) or levels are easily influenced by other factors (e.g. cortisol varies markedly with stress levels and has a marked circadian rhythm; see Table 2.1).

In general

•If you are suspecting a LOW level—do a STIMULATION test (to see if it stays LOW)

•If you are suspecting a HIGH level—do a SUPPRESSION test (to see if it stays HIGH)

2.Use the correct collection method, e.g. ACTH or insulin levels require rapid separation of the sample and prompt freezing (−20°C). Timing of sampling may also be critical. Label samples carefully, including time of collection! Check procedures with the local laboratory. Many units will have protocols for endocrine investigations.

3.Do tests in the correct sequence, e.g. ACTH levels can only be interpreted once the cortisol status is known. In many cases, simultaneous samples are required for interpretation, e.g. PTH with Ca²⁺ for hypo-/hyperparathyroidism, glucose with insulin for insulinoma.

4.‘Normal’ results may be ‘abnormal’, depending on the activity of the hormone axis under investigation. Interpretation of the absolute levels of hormones in isolation may be highly misleading. For example, a serum PTH within the normal range in the presence of hypocalcaemia suggests hypoparathyroidism; ‘normal’ LH and FSH levels in the presence of a very low serum testosterone concentration suggest pituitary failure. In both instances, the regulatory hormone concentration is inappropriately low. Thus, the level of the regulatory hormone (or releasing factor) must be considered in the light of the simultaneous level of the ‘target’ hormone or metabolite.

5.Results may vary according to the laboratory assay. Reference ranges vary between laboratories—it is especially important with endocrine tests to interpret your results according to your laboratory’s ‘normal range’. Also, interfering factors may differ between assays, e.g. different PRL assays cross-react very differently with macroprolactin ( Galactorrhoea (hyperprolactinaemia), pp. 172–173). Some individuals have a heterophile interfering antibody that affects the results of many radioimmunoassays. Resist ‘discarding clinical evidence in favour of a numerical value’.

6.Beware of interfering medication, e.g. inhaled beclomethasone can suppress serum cortisol levels; administered hydrocortisone (cortisol) is detected by the cortisol assay; synthetic androgens and oestrogens can appear to cause low serum testosterone/oestrogen (as they are not detected in the testosterone/oestrogen assay); some anti-emetics and antipsychotics can raise circulating PRL levels; carbenoxolone or liquorice may cause hypokalaemia. Always ask patients for a full medication list (including herbal remedies and other self-medications).

7.Take a family history. Familial forms of many common endocrine problems exist that require important changes in the management approach, e.g. familial hypercalcaemia may suggest MEN-1 or MEN-2 requiring a different form of parathyroid surgery and a risk of phaeochromocytoma (MEN-2).

Endocrine tests are generally expensive and should not be performed unnecessarily or outside standard protocols. Dynamic tests may have cautions and contraindications and can be hazardous if used inappropriately (see Table 2.1). A high degree of organization and close liaison with the laboratory are required to perform these tests in a way that can be clearly interpreted. Dynamic tests should ideally be performed in an endocrine investigation unit. Chemical pathologists (clinical biochemists) and other laboratory staff generally have great experience with performing and interpreting endocrine tests—seek their advice, wherever possible, before embarking on tests with which you are unfamiliar. Tests in children should be performed and interpreted under expert paediatric guidance.

Table 2.1 Random sampling vs dynamic testing

Hormone	Random or dynamic sampling?
GH	Dynamic: glucose tolerance test for excess; insulin stress test or GHRH arginine stimulation test for deficiency
IGF-1	Random
LH, FSH	Random in ♂, post-menopausal Timed with menstrual cycle in premenopausal ♂ Random or stimulated in pre-pubertal children
Testosterone	Random
Oestrogen (oestradiol)	Random in ♂, post-menopausal ♂ Timed with menstrual cycle in premenopausal
ACTH	Random
Cortisol	Dynamic: dexamethasone suppression test for excess; Synacthen^® stimulation test if suspect deficiency
TSH	Random
T4 and T3	Random
PRL	Random
ADH/vasopressin	Do not normally measure directly
Osmolality	Dynamic: water deprivation test if suspect DI
PTH	Random, but need simultaneous Ca²⁺ value
Insulin	Fasting, plus simultaneous glucose value
Calcitonin	Random
Renin/aldosterone	Upright usually, off medication
Metanephrines	Measure in urine, 24h sample or seated random plasma sample
5HIAAs	Measure in urine, 24h sample

Further reading

American Association of Clinical Endocrinologists Clinical Guidelines.AACE/ACE clinical practice guidelines. https://www.aace.com/publications/guidelines.

Endocrine Society. Endocrine Society Guidelines. https://www.endocrine.org/guidelines-and-clinical-practice.

Ismail AA, Barth JH. Wrong biochemistry results. BMJ 2001; 232: 705–6.

Hypothalamus/pituitary function

Hypothalamic dysfunction

Hypopituitarism

Definition

Failure of one or more pituitary hormones (usually multiple).

Causes

•Congenital.

•Pituitary tumour (including infarction of tumour ‘apoplexy’).

•Craniopharyngioma.

•Post-cranial irradiation.

•Following pituitary irradiation.

•Metastases to the pituitary (especially the breast).

•Post-surgery.

•Empty sella syndrome (occasionally).

•Sheehan’s syndrome (infarction with postpartum haemorrhage).

•CTLA-4 Ig immunotherapy for malignancy.

Symptoms and signs

Often very vague, e.g. tiredness, normocytic anaemia (easily missed). Combined with impotence or amenorrhoea—very suggestive. Other clues include loss of body hair (especially axillary), reduced shaving, hyponatraemia, and growth failure in children. DI is not a feature (unless there is also hypothalamic damage), as ADH can be secreted directly from the hypothalamus. There may also be signs of an SOL—bitemporal hemianopia (rarely optic nerve compression, homonymous hemianopia); headache (especially following apoplexy); III, IV, V1, V2, or VI cranial nerve lesions; and CSF rhinorrhoea. Occasionally, galactorrhoea following pituitary stalk compression by a tumour (‘disconnection’). Note: generally GH is lost first, then LH/ FSH, and ACTH/TSH last. If multiple pituitary hormones are deficient, GH deficiency can be assumed.

Investigations

(See Fig. 2.1.) Basal-free T4 (not TSH, which can be misleadingly normal), LH, FSH, PRL, and oestrogen/testosterone are usually sufficient to test the thyroid and gonadal axes. Adrenal and growth hormone testing requires dynamic tests (e.g. insulin tolerance test (ITT), but see below). Severe GH deficiency = peak stimulated GH <12.3mU/L (4.1ng/mL) using GHRH arginine test; <15.3mU/L (5.1ng/mL) using ITT testing. Note that the short Synacthen^® test ( Short Synacthen^® test, p. 225) is only suitable for testing the hypothalamopituitary–adrenal axis if pituitary failure is long-standing (>6 weeks), allowing time for adrenal atrophy to occur.

Alternative investigations

•GHRH arginine stimulation test. Although the ITT ( Test protocols, pp. 216–217) is the traditional gold standard test for GH and 2° adrenal insufficiency, the GHRH arginine test has equal sensitivity and specificity for GH deficiency. It is also safer and better tolerated and is gradually replacing the ITT.¹

•Combined anterior pituitary testing—giving LHRH, thyrotropin-releasing hormone (TRH), ACTH, and GHRH ( Combined anterior pituitary function testing, p. 218)—no clear advantage of this approach has been demonstrated and the results of the LHRH test, in particular, are difficult to interpret in pre-pubertal children.

•Long (depot) Synacthen^® test—rarely required ( Adrenal failure, pp. 162–164; Long (depot) ACTH test, p. 225).

Fig. 2.1 Investigation of suspected hypopituitarism. Note: specialist paediatric advice should be taken in children.

Further reading

Molitch ME, Clemmons DR, Malozowski S, Merriam GR, Vance ML; Endocrine Society. Evaluation and treatment of adult growth hormone deficiency. J Clin Endocrinol Metab 2011; 96: 1587–609.

Acromegaly (growth hormone excess)

For GH deficiency, see Hypothalamus/pituitary function, pp. 128–130.

Clinical features

•Often insidious over many years.

•Enlarging hands and feet with rings having to be resized.

•Increase in shoe size.

•Coarsening of facial features, especially enlargement and broadening of the nose.

•Sweating.

•Headache.

•Malocclusion (protuberance of the lower jaw) and splaying of the teeth.

•Carpal tunnel syndrome.

•DM.

•May be local symptoms from the pituitary tumour and symptoms/signs of loss of other pituitary hormones ( Hypothalamus/pituitary function, pp. 128–130).

•GH excess commencing before puberty results in gigantism (tall stature).

Investigations

•A random GH level is not helpful—may be high in normal people.

•A random IGF-1 level should be measured and compared with laboratory normal ranges corrected for age. This can be used as a screening test, but IGF-1 assays vary in reliability. IGF-1 levels should be raised in all cases of acromegaly, but levels can be affected (reduced) by fasting and systemic illness.

•Where IGF-1 results suggest GH excess, this should be confirmed in a standard 75g OGTT with glucose and GH measurements at 0, 30, 60, 90, and 120min.

•If no GH values are <2mU/L, then a diagnosis of acromegaly is confirmed.

•The vast majority (99%) of cases of acromegaly are due to pituitary tumours. If a pituitary tumour is not seen on MRI scanning, yet acromegaly is confirmed, a GHRH level should be requested to exclude ectopic production of this polypeptide by non-pituitary tumours stimulating the release of GH from the pituitary.

•For follow-up of treated cases of acromegaly, IGF-1 levels (more sensitive) and/or random GH levels can be used.

•Life expectancy appears to return to normality when the nadir of GH values is <3mU/L or IGF-1 levels return to the reference range.

Further reading

Katznelson L, Laws ER Jr, Melmed S, et al.; Endocrine Society. Acromegaly: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2014; 99: 3933–51.

Polydipsia and polyuria: diabetes insipidus

‘First-line tests’

It is relatively common for patients to report excess thirst or ↑ need to pass urine. Figure 2.2 and Box 2.1 summarize the causes. Prostatism and urge incontinence resulting in urinary frequency should be distinguished by history-taking as the patients do not have thirst. Then the first step is to identify straightforward causes, such as drugs (diuretics), DM, hypercalcaemia, hypokalaemia, and chronic renal failure with U&E, creatinine, glucose, and Ca²⁺. Measuring 24h urine volume is also useful as volumes over 3L are likely to be pathological and volumes under 2L do not require further investigation. A GTT should not be required to diagnose DM as the renal threshold for glucose needs to be exceeded (~10mmol/L) to cause polyuria and there should be glucose in the urine.

Box 2.1 Causes of polyuria/polydipsia

Diabetes mellitus

•DI (cranial or nephrogenic)

•High Ca²⁺

•Low K⁺

•Chronic renal failure

•1° polydipsia (including dry mouth, e.g. Sjögren’s)

‘Second-line tests’

Once other diagnoses have been excluded, subsequent tests aim to distinguish DI from 1° polydipsia (compulsive water drinking). A carefully supervised water deprivation test should be performed ( Water deprivation test, pp. 220–221). However, it is not always easy to arrive at a conclusive diagnosis. Serum Na⁺ levels are helpful, as DI is unlikely if Na⁺ <140mmol/L. Morning spot urine osmolality after overnight water restriction (not shown on chart) is occasionally useful—values >600mOsmol/L make significant degrees of DI unlikely. Measuring 24h urine volume is also useful, as volumes over 3L are likely to be pathological. However, obligate urine volumes as low as 2L could still cause the patient to complain of polyuria. In such borderline cases, the distinction between partial DI, normality, and 1° polydipsia can be very difficult. It has been proposed that in an adult or child over 2 years of age, a 24h urine volume of >40mL/kg body weight, a plasma osmolarity of <300mOsmol/L, and a −ve test for glucose is diagnostic of DI, but this requires confirmation.

Guidance on interpretation of second-line tests, including the water deprivation test, is given in Table 2.2. Note that 1° polydipsia may be a psychiatric condition but can also occur in patients with a dry mouth (e.g. Sjögren’s syndrome, anticholinergic drugs) or who have been previously encouraged to drink regularly ‘to help their kidneys’.

Distinction between partial cranial DI and habitual (psychogenic) water drinking is complicated by the fact that drinking very high volumes over time may ‘wash out’ the renal medullary concentrating gradient. In this situation, a plasma vasopressin level at the end of the water deprivation test may be very helpful to distinguish a lack of vasopressin from a lack of vasopressin action. A 24h urine volume is also helpful as volumes of <3L/day are unlikely to cause renal ‘washout’. Clues to 1° polydipsia include an initial plasma osmolality (and serum Na⁺) that is low, plasma osmolality rising to >295mOsmol/L, and thirst not abolished by desmopressin, despite a rise in urine osmolality. Note that ‘full-blown’ cranial DI results in urine volumes of around 500mL/h (12L/day).

Fig. 2.2 Investigation of polydipsia/polyuria. Note: once cranial DI is diagnosed, further investigations into the underlying cause are required ( Hypothalamic dysfunction under Hypothalamus/pituitary function, p. 128).

Interpretation of second-line tests for polyuria/polydipsia

Table 2.2 Interpretation of second-line tests for polyuria/polydipsia

	Normal	Partial DI: cranial (C) or nephrogenic (N)	Primary polydipsia
Random serum Na⁺	Normal	>140mmol/L	<140mmol/L
Random serum osmolality**	Variable	>290	<290
Morning urine mOsm**	Variable	Unlikely if >600 (C) Excluded if >600 (N)
End of water deprivation test before desmopressin**	Urine >600 Plasma 280–295	Urine <600 Plasma >295	Urine >600 * Plasma 280–295
Urine osmolality after desmopressin SC**	>600	Rises to >600 or >50% ↑ (C); rises to <600 or <50% ↑ (N)	Rises to >600* or >50% ↑
Plasma vasopressin at end of water deprivation test	Normal for plasma osmolality	Low for plasma osmolality (C); normal for plasma osmolality (N)	Normal for plasma osmolality

* With long-standing large-volume polyuria (>3L/day), these values may not be achieved due to washout of the renal medullary concentrating gradient—if results equivocal, see text.

** Osmolalities are all expressed in mOsmol/L.

‘If all else fails’

In cases of doubt, a carefully supervised therapeutic trial of desmopressin can be useful to distinguish DI from 1° polydipsia ( Diagnostic trial of desmopressin, p. 222). This should be done as an inpatient, as there is a risk of significant hyponatraemia in habitual water drinkers. The principle is that patients able to regulate water intake according to their thirst (DI) should not develop a hypo-osmolar plasma. In 1° polydipsia, the urine volume will fall and the urine concentrating gradient will gradually recover. However, if the patient continues to drink due to their psychological drive, rather than their thirst, they will become water-overloaded and hypo-osmolar.

An additional valuable test to distinguish partial DI from 1° polydipsia is hypertonic saline infusion testing, which usually requires access to a plasma vasopressin assay but has been used with urinary vasopressin levels.²–³ MRI scanning typically shows an ↑ signal in the posterior pituitary, which is lost in cranial DI. However, this sign is not helpful in distinguishing more subtle degrees of DI from other causes.

Hyponatraemia (including syndrome of inappropriate antidiuretic hormone)

Hyponatraemia is a very common clinical problem. Figure 2.3 shows a flow chart for investigation. If patients are on diuretics, further evaluation is usually not possible. The diuretic will need to be discontinued. If this is not possible, hyponatraemia is likely to be attributable to an underlying condition (cardiac, renal, or liver failure). Pseudo- or dilutional hyponatraemia is important to exclude at an early stage (see Box 2.2). A careful clinical assessment should be made of the volume status, including identification of oedema, fluid loss (e.g. diarrhoea, fistula leakage), and signs of dehydration, including postural drop in BP. A urine Na⁺ and TSH estimation is useful at this stage (see Fig. 2.3). Note that the most important diagnosis not to miss is hypoadrenalism, as this can be fatal if untreated. Clinicians should have a low threshold for performing a short Synacthen^® test ( Short Synacthen^® test, p. 225). Hypoadrenalism due to pituitary failure may not be accompanied by hyperkalaemia, hypotension, or hyperpigmentation and can easily be missed. Cerebral salt wasting occurs within days of brain injury (e.g. SAH, neurosurgery, or stroke) and is probably due to release of brain natriuretic peptides.

The syndrome of inappropriate antidiuretic hormone (SIADH) is a diagnosis of exclusion (see Box 2.3).

All the criteria in Box 2.3 should be met. A specific cause for SIADH is frequently not found or there may be a combination of precipitating factors (see Table 2.3). In the elderly, a state of chronic SIADH is relatively common and usually explains hyponatraemia persisting for many years without any other apparent cause. Affected individuals should be encouraged to drink less than a litre a day (‘5 cups or less’), to only drink if they are thirsty, and to avoid exacerbating factors (see Table 2.3).

Table 2.3 Causes of SIADH

Cause	Examples
Drugs	Carbamazepine, chlorpropamide, opiates, psychotropics, cytotoxics
CNS disorders	Head trauma, post-pituitary surgery (transient), stroke, cerebral haemorrhage, GBS, meningitis, encephalitis, fits
Malignancy	Small-cell lung cancer, pancreas, prostate
Chest disease	Pneumonia, TB, abscess, aspergillosis
General stimuli	Nausea, pain, smoking
Other	Acute intermittent porphyria

Features of SIADH/hyponatraemia that are often underappreciated

1.Other than a CXR, there is no requirement to search for an underlying malignant cause. If there is underlying malignancy, it is usually extensive, very apparent, and incurable (e.g. extensive small-cell carcinoma of the lung).

2.The urine osmolality does not have to be high. In individuals drinking large volumes of fluid, a urine osmolality as low as 250mOsmol/L (i.e. less than plasma) may be inappropriately concentrated, reflecting true SIADH.

3.Conditions previously diagnosed as ‘sick cell syndrome’ are now thought to represent SIADH in ill patients.

4.‘Water intoxication’ is usually the combination of SIADH and excessive fluid intake. Healthy patients drinking to excess can rarely exceed the renal capacity to excrete a water load (~12L/day) and hence do not become hyponatraemic. A degree of SIADH is required for potomaniacs (excess water drinkers) to become hyponatraemic.

5.The post-operative state contains many precipitants to SIADH (nausea, pain, opiates, pneumonia) and ADH secretion is promoted by hypovolaemia from blood loss. The administration of ‘3L of IV fluid a day’ post-operatively frequently results in hyponatraemia.

6.Symptoms of hyponatraemia, such as drowsiness, coma, or fits, are dependent on the rate of fall of serum Na⁺, not the absolute value. Patients who are alert with Na⁺ <125mmol/L have clearly been chronically hyponatraemic and their serum Na⁺ requires only gentle correction. However, a very rapid fall in serum Na⁺ to <130mmol/L (typically due to massive infusion of hypotonic fluid into the bladder) may cause coma and needs to be corrected as a medical emergency with hypertonic saline.

Fig. 2.3 Investigation of hyponatraemia.

Further reading

Spasovski G, Vanholder R, Allolio B, et al.; Hyponatraemia Guideline Development Group. Clinical practice guideline on diagnosis and treatment of hyponatraemia. Eur J Endocrinol 2014; 170: G1–47.

Obesity/hypercortisolism

Endocrinologists are frequently asked to determine whether there is an underlying endocrine cause in patients who are obese. 2° causes of obesity are listed in Box 2.4. A long history of obesity, typically going back to childhood, is characteristic of constitutional obesity and further investigation, other than thyroid function, is rarely necessary. However, simple obesity may result in effects suggestive of hypercortisolism, e.g. striae, bruising, central obesity, rounded facial features, mild hyperandrogenism in women, buffalo hump, hypertension, and hyperglycaemia. Rapidly progressive obesity, marked hypertension, hypokalaemia, proximal muscle weakness, poor sleep, osteoporosis/vertebral collapse, and marked hirsutism or acne are more suggestive of hypercortisolism and require further investigation. Hypothalamic damage is usually apparent from the history.

The optimal approach to the diagnosis of hypercortisolism (Cushing’s syndrome) is probably the most controversial subject in endocrinology. Endocrinologists who have seen many cases of Cushing’s syndrome have seen exceptions to every rule, and the episodic nature of ACTH and cortisol secretion means that low values can occur even in disease. True cyclical Cushing’s disease also occurs but is rare.

Diagnosis consists of two phases

1.Does the patient have hypercortisolism or not?

2.What is the cause of the hypercortisolism? Phase 1 must be completed first, as phase 2 tests can only be interpreted if hypercortisolism is present.

Investigation of hypercortisolism phase 1

Does the patient have hypercortisolism?

Patients being investigated for hypercortisolism should look Cushingoid. One exception is malignant ectopic ACTH secretion (e.g. from small-cell lung cancer) which can cause profound hypokalaemia before Cushingoid appearance is apparent. Depression and alcoholism may cause abnormal tests for hypercortisolism without representing a true hypercortisolaemic state and hence are termed ‘pseudo-Cushing’s syndrome’. Such depressed patients often do not appear Cushingoid and alcoholism should be identifiable clinically and biochemically. If there is a high degree of suspicion of hypercortisolism in a depressed patient, midnight cortisol levels <140nmol/L or a −ve result on dexamethasone–corticotropin-releasing hormone (CRH) testing ( Low-dose dexamethasone suppression test, p. 223) may be helpful in excluding the diagnosis. Note that iatrogenic or factitious Cushing’s syndrome is usually due to a steroid other than hydrocortisone (not detected in the cortisol assay) and characteristically results in an apparent suppression of the hypothalamopituitary–adrenal axis on testing.

Four tests are used to determine whether a patient does have hypercortisolism

1.24h urinary-free cortisol (UFC) collections. Three collections with simultaneous creatinine excretion estimation are ideal. If the creatinine excretion varies by >10% between collections, the samples are not true 24h collections and should be repeated. If two or more collections have a value >3 times the laboratory upper limit of normal (e.g. >800nmol/24h), then the diagnosis of hypercortisolism is secure. Patients with intermediate values should have repeat sampling after several weeks or additional tests. Steroids, adrenal enzyme inhibitors, statins, and carbamazepine must be discontinued prior to testing. False +ves can be caused by pregnancy, anorexia, exercise, psychoses, alcohol, and alcohol withdrawal.

2.Low-dose dexamethasone suppression test (LDST). This can be performed overnight or over 2 days ( Low-dose dexamethasone suppression test, p. 223), the latter having less false +ves. Some authorities believe it adds little to UFCs as when cortisol secretion is high, the UFC is clearly raised, but in times when it is intermediate, the LDST may be normal. It is a useful outpatient screening test ( Overnight dexamethasone suppression test under Low-dose dexamethasone suppression test, p. 223) in individuals who cannot reliably collect 24h urine samples.

3.Late-night salivary cortisol test. High salivary cortisol levels (>4nmol/L) measured between 11 p.m. and 12 midnight indicate loss of diurnal rhythm and are one of the best tests of hypercortisolism. Salivary samples can be collected as an outpatient by drooling into a collection tube or use of a cotton pledgelet. The tests should be repeated on two occasions. An alternative is a venous sample taken via an indwelling cannula in as relaxed a state as possible, preferably during sleep, but this requires an inpatient admission. Values <140nmol/L make hypercortisolism very unlikely.

4.Dexamethasone-suppressed CRH test ( Low-dose dexamethasone suppression test, p. 223). This is a modification of the LDST, which was initially claimed to have a specificity of 100% for hypercortisolism, but subsequent series suggest <80%.

Summary

In patients who appear Cushingoid, three UFCs should be performed (note causes of false +ves). If these give equivocal results, additional tests are required, including further UFCs, late-night salivary cortisols, and a formal 2-day LDST followed by CRH.

Investigation of hypercortisolism phase 2

What is the cause of hypercortisolism?

The common and rare causes of hypercortisolism are summarized in Tables 2.4 and 2.5, along with useful clinical features. ~65% of cases are due to a pituitary adenoma (Cushing’s disease), 20% due to an adrenal adenoma or carcinoma, and 10% due to ectopic ACTH production.

These are the three main causes to be distinguished using a combination of the tests shown under Investigations below. Distinction between a pituitary adenoma (which may not be visualizable on MRI) and a small indolent tumour (typically lung carcinoid) represents the greatest challenge. Despite extensive investigation, the cause will remain uncertain in some of these cases.

Investigations

1.Plasma ACTH level (separate and freeze immediately). Undetectable plasma ACTH levels are strongly suggestive of an adrenal tumour. However, ACTH secretion is intermittent and two suppressed values with simultaneous high cortisol levels (>400nmol/L) are preferable and should prompt adrenal CT scanning.

2.High-dose dexamethasone suppression test ( High-dose dexamethasone suppression test, p. 224). Greater than 90% suppression of basal UFC levels is strongly suggestive of a pituitary adenoma. Lesser degrees of suppression are seen with ectopic ACTH.

3.Inferior petrosal sinus sampling (IPSS). This is an excellent diagnostic tool but requires expert radiological support and should only be performed in tertiary referral centres. A total of 100mg IV of CRH is also given via a peripheral vein, while sampling, to ensure active secretion of ACTH during the test. ACTH levels are compared between the inferior petrosal sinus on both sides and a peripheral vein. Sampling is performed at −15, 0, +15, and +30min after CRH injection. Ratios >2 (ideally >3) post-CRH are strongly suggestive of pituitary-dependent disease. Risks include failure to enter the sinus and sinus thrombosis.

4.Imaging. Pituitary and adrenal imaging should not be performed without biochemical testing, as non-functioning tumours of the pituitary and adrenal are common (false +ves), and conversely functioning pituitary tumours are often too small to be visualized by MRI (false −ve). However, if the findings are consistent with the biochemical tests, this is useful supportive evidence. Patients with findings suggestive of ectopic ACTH production should have thin-slice CT of the chest looking for a bronchial adenoma, and MRI scanning of the pancreas for an islet tumour. ¹¹¹Indium-labelled octreotide scanning may also be useful in locating small tumours.

5.Plasma CRH levels. Very rarely, ‘ectopic ACTH’ syndrome is actually due to ectopic CRH production stimulating ACTH from the pituitary (see Table 2.5). Raised plasma CRH levels may be diagnostic in this condition.

Additional tests include

•Metyrapone test: here the adrenal enzyme blocker metyrapone is used to lower cortisol levels. Pituitary adenomas respond by ↑ ACTH production, but ectopic sources of ACTH do not. The test can also be used to confirm that ACTH levels are truly suppressed in adrenal tumours (rarely necessary).

•Peripheral CRH test: ACTH levels are measured before (−30, −15min) and +15 and +30min after injection of 100mg IV of CRH into a peripheral vein. A rise in ACTH levels of >34% is suggestive of a pituitary adenoma. The addition of 5µg IV of desmopressin improves the response rate and reduces false −ves.

Table 2.4 Common causes of hypercortisolism (Cushing’s syndrome)

Cause	Pathology	Characteristic features
ACTH-secreting pituitary adenoma (Cushing’s disease)—65%	Pituitary adenoma	Typical features of hypercortisolism with little virilization
Ectopic ACTH secretion—10%	Malignant: small-cell lung cancer, thymic carcinoid, medullary thyroid cancer Indolent/benign: bronchial/pancreatic carcinoids, phaeochromocytoma	Malignant: rapid progression, marked hypokalaemia, proximal muscle weakness, ↑ BP, tumour clinically apparent, few Cushingoid signs Indolent: indistinguishable from Cushing’s disease, tumour not easily detected
Adrenal tumour—20%	Adrenal adenoma Adrenal carcinoma	Adenomas: typical Cushingoid signs, sometimes virilization Carcinomas: rapid progression (months) with virilization, poor prognosis

Summary

(See Fig. 2.4.)

Fig. 2.4 Hypercortisolism. Flow chart for diagnosing the cause once hypercortisolism is established.

Table 2.5 Rare causes of hypercortisolism (Cushing’s syndrome)

Cause	Pathology	Characteristic features
Ectopic CRH secretion	Variety of tumours, mostly carcinoids	Clinical features indistinguishable from Cushing’s disease, but no pituitary tumour, ↑ serum CRH and may fail to suppress with high-dose dexamethasone
Ectopic gastrin-releasing peptide secretion	Medullary thyroid cancer	Very rare, resembles ectopic CRH
Factitious ACTH administration	Injections of ACTH	Very difficult to distinguish from ectopic CRH secretion or Cushing’s disease but, if isolated from their ACTH source, become adrenally insufficient in days
Cyclical Cushing’s disease	Cyclical secretion from pituitary adenoma	Cushing’s disease with intermittently −ve tests
Pseudo-Cushing’s syndromes	Depression or alcoholism	Clinical evidence of Cushing’s disease may be limited; evidence of depression or alcoholism
Bilateral micronodular adrenal hyperplasia	Often associated with Carney complex	Investigation suggestive of adrenal tumour (ACTH suppressed), but adrenals normal or slightly enlarged and contain pigmented nodules
Bilateral macronodular adrenal hyperplasia	Sporadic or familial	Investigation suggestive of adrenal tumour (ACTH suppressed), but marked or very marked bilateral nodular enlargement of adrenals on CT scanning

Further reading

Nieman LK, Biller BM, Findling JW, et al. The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2008; 93: 1526–40.

Endocrine hypertension

Ninety-five per cent of cases of hypertension are ‘essential hypertension’ with no specific underlying cause. If hypertension is very marked, occurs in younger patients, is difficult to control with drugs, is episodic/fluctuating, is of recent onset, is familial, is associated with recurrent hypokalaemia, or has associated features (see Table 2.6), then an underlying cause should be excluded.

History and examination should include features of conditions in Table 2.6, with particular attention to paroxysmal attacks, drugs (e.g. liquorice), and family history.

Table 2.6 Secondary causes of hypertension

Physical features	Notes
Absent
Phaeochromocytoma	May be familial, e.g. in MEN-2 (may have mucosal neuromas), von Hippel–Lindau syndrome, neurofibromatosis; paroxysmal ↑ BP in only 60% of cases with headache, sweating, and palpitations
Hyperaldosteronism	Multiple syndromes, including Conn’s syndrome (see Table 2.7)
Renal artery stenosis	Congenital or acquired (atheroma)
Renal disease	Any cause, including polycystic kidneys
Hyper-/hypothyroidism	Diastolic hypertension with hypothyroidism, systolic hypertension with hyperthyroidism
Hyperparathyroidism	Does not usually improve after surgical cure
Drugs	Epo, ciclosporin, cocaine, amphetamines, steroids, liquorice, oestrogens, and androgens
Physical features present
Coarctation of the aorta
Cushing’s syndrome
Acromegaly
Pregnancy-induced

Figure 2.5 provides a flow chart for further investigations. At least three separate BP readings should be obtained—24h BP monitoring may be useful where ‘white coat hypertension’ is suspected.

Fig. 2.5 Investigation of causes of hypertension.

The majority of 2° causes of hypertension can be rapidly excluded by the investigations shown in the first box of Fig. 2.5. If the results are normal or the only abnormality is a low K⁺ level, then the possibilities of hyperaldosteronism or RAS remain to be distinguished from essential hypertension. Further investigation should be driven by the severity of the hypertension, the (young) age of the patient, and the difficulty in obtaining control with drugs.

Investigation of renal artery stenosis/high renin levels

Selective renal angiography remains the gold standard for diagnosing RAS—other imaging methods can miss the diagnosis. Three-dimensional (3D) MR angiography is now considered a non-invasive alternative. High renin levels associated with hypertension (off drugs) in the absence of RAS should prompt a search for a juxtaglomerular cell tumour of one kidney. Note that the presence of hypertension is essential, as many conditions associated with a low or normal BP can result in ‘appropriate’ hyper-reninaemia (e.g. diuretics; cardiac, renal, or liver failure; hypocortisolism; hypovolaemia). High renin levels can also occur in essential hypertension.

Investigation of hyperaldosteronism

Hypertension with persistent hypokalaemia raises the possibility of hyperaldosteronism which may be due to a variety of causes (see Table 2.7). Note that investigation for hyperaldosteronism is also appropriate with K⁺ levels in the normal range if other investigations are −ve and hypertension is marked, difficult to control, or in a younger patient. The optimal approach to investigation remains controversial and equivocal cases frequently occur. If there is marked hypokalaemia of recent onset, a 24h UFC (and review of medication) is indicated to exclude recent-onset hypercortisolism (usually due to ectopic ACTH production) in which Cushingoid features have not yet become apparent. True hyperaldosteronism is never due to a malignant lesion, so that if hypertension can be medically controlled, it is not always necessary to establish a definitive diagnosis of aetiology. A detailed scheme is provided in Fig. 2.6.

Establishing hyperaldosteronism

The initial investigation is an upright aldosterone/renin ratio, performed when the patient has been upright or sitting (not lying) for at least 2h. The sample needs to be taken to the laboratory, separated, and frozen immediately. Ideally, the patient should be on no antihypertensives other than α-blockers (e.g. doxazosin), as most drugs can affect interpretation of the test results (see Table 2.8). This is difficult to achieve in subjects with very marked hypertension. Combination antihypertensive therapy and spironolactone cause the most confusion. An undetectable renin with an unequivocally high aldosterone level makes the diagnosis very likely. A normal or raised upright renin excludes hyperaldosteronism. Borderline results should be repeated off interfering medication and after K⁺ replacement (hypokalaemia can inappropriately lower aldosterone). A low renin with a normal aldosterone can be seen in essential (‘low renin’) hypertension. Refer to the laboratory for normal and diagnostic ranges. Additional tests (e.g. renin after Na⁺ restriction/furosemide, aldosterone after captopril, Na⁺ loading, or IV saline) are used in specialist centres, but their exact role in testing remains unresolved.

Table 2.7 Investigating established primary hyperaldosteronism

	Change in aldosterone with posture	CT findings	Adrenal venous sampling (ratio of aldosterone between sides)	Response to glucocorticoids*	Treatment of choice	Notes
Adenoma (Conn’s)	None/fall	Unilateral nodule	>10:1	Absent	Surgery
Renin-responsive adenoma	Rise	Unilateral	>10:1	Absent	Surgery
Unilateral hyperplasia	None/fall	‘Normal’	>10:1	Absent	Surgery
Bilateral hyperplasia	Rise	‘Normal’	No difference	Absent	Medical
Glucocorticoid remediable aldosteronism (GRA)	None/fall	Normal	No difference	Present	Steroids	Very raised 18-oxo cortisols. Positive genetic screening**

* Dexamethasone 0.5mg 6-h for 2–4 days resulting in supperession of aldosterone levels to nearly undetectable levels (usually associated fall in BP also).

** Positive for chimeric CYP11B1/CYP11B2 gene.

Fig. 2.6 Investigation of hyperaldosteronism/mineralocorticoid excess in patients with hypertension.

Table 2.8 Renin/aldosterone testing and drugs

Drug	Effect on PRA	Effect on aldosterone
Drugs that ↑ PRA
Spironolactone	↑	Variable
Ca²⁺ channel blockers	May ↑	↓
ACE inhibitors*	↑	↓
Diuretics	↑	↑
Vasodilators	↑	↑
Drugs that ↓ PRA	↓	↓
α-blockers	↓	↓
NSAIDs

* Angiotensin II receptor antagonists are likely to have same effects.

PRA, plasma renin activity.

Investigating the cause of established primary hyperaldosteronism

There are five causes of established 1° hyperaldosteronism with suppressed renin and high aldosterone (see Box 2.5). Surgery (unilateral adrenalectomy) is indicated for adenoma (65% of cases), the unusual renin-responsive adenoma, and the rare cases of unilateral hyperplasia, but not for bilateral hyperplasia (idiopathic hyperaldosteronism, 30% of cases) or the rare familial glucocorticoid-remediable aldosteronism (GRA). Tests to distinguish these are summarized in Box 2.5 and Fig. 2.7.

OHCM 10e, pp. 228–9.

Fig. 2.7 Identifying the cause of established primary hyperaldosteronism.

If hyperaldosteronism is established and a nodule is visible on CT/MRI imaging, it is reasonable to proceed to unilateral adrenalectomy/excision of the nodule. If no nodule or bilateral nodules are seen, then adrenal vein sampling is the most useful test to determine whether surgery should be performed. Aldosterone levels after glucocorticoid administration or genetic testing for the chimeric CYP11B1/CYP11B2 gene should be performed beforehand to exclude GRA (see Table 2.7, p. 151—family members may be only mildly hypertensive, making family histories unreliable). Unfortunately, the right adrenal vein cannot be catheterized in up to 25% of cases and there is a risk of precipitating adrenal haemorrhage. Postural studies identifying a >50% rise in aldosterone comparing recumbent and 2–4h of standing/walking suggest idiopathic hyperplasia, but a small renin-responsive adenoma not visible on CT could give similar results.

Investigating the cause of apparent mineralocorticoid excess

Rarely, investigation reveals low renin and low aldosterone levels in the presence of hypertension, hypokalaemia, and alkalosis. There are five causes of this (see Box 2.5). A 24h UFC estimation will rapidly exclude recent-onset, aggressive hypercortisolism. Repeated enquiry should be made for drug and liquorice product ingestion. The remaining causes may be diagnosed by urinary cortisol/cortisone ratio (11β-OH steroid dehydrogenase deficiency—often referred to alone as ‘apparent mineralocorticoid excess’) or other appropriate changes in urinary and plasma cortisol metabolites (e.g. raised DOC levels—11β-hydroxylase or 17α-hydroxylase deficiency) or responsiveness to amiloride (Liddle’s syndrome).

Phaeochromocytoma

1.Clinical features. Phaeochromocytoma is rare, but an important diagnosis not to miss—can result in fatal hypertensive crisis, especially during surgery or after inadvertent β-adrenoreceptor blockade without α blockade. It can be sporadic (90%) or be the first clue to a familial syndrome (see Table 2.6). ~10% of cases are extra-adrenal, 10% multiple, and 10% malignant (‘tumour of 10%’). Ninety per cent of cases have sustained or paroxysmal hypertension, but paroxysmal attacks of some nature are a feature of only 55% of cases. Pure adrenaline-secreting lesions can occasionally cause hypotension. They are always intra-adrenal. Phaeochromocytoma needs to be excluded in cases of incidentally found adrenal masses. Paragangliomas are non-secreting phaeos.

2.Diagnostic tests. Plasma free or 24h urinary fractionated metanephrines have replaced catecholamine estimations or catecholamine metabolites (vanillylmandelic acids (VMAs)), as they are more sensitive and specific and are released continuously. A single clearly positive estimation in the presence of hypertension is usually sufficient. If non-diagnostic, sampling in the recumbent position may help confirm normal levels (metanephrines are 2-fold higher in the seated position). Mild ↑ can be seen in anxiety states and with very small lesions detected in the follow-up of familial, recurrent disease. Causes of false +ve results include methyldopa, levodopa, labetalol, sotalol, tricyclic and monoamine oxidase inhibitor (MAOI) antidepressants, paracetamol, sulfasalazine, sympathomimetics, cocaine, clonidine withdrawal, intracranial events (e.g. SAH, posterior fossa tumour), or metabolic stress (e.g. hypoglycaemia, MI).

3.Finding the tumour. Once the diagnosis is established, blockade (typically with increasing twice-daily (bd) doses of phenoxybenzamine) should be established before invasive investigation. The tumours are usually large (>2cm) and bright on T2-weighted (T2W) images. CT/MRI scanning therefore identifies virtually all adrenal lesions. Radionuclide scanning with ¹³¹I-MIBG (iodine-131-meta-iodobenzylguanide) is useful to confirm activity if >1 adrenal nodule is present and to identify extra-adrenal lesions where no adrenal lesion is seen. Note that extra-adrenal phaeochromocytomas (paragangliomas) are usually in the chest or abdomen but can occur in the neck (including chemodoctomas of the carotid body), pelvis, and bladder. Biopsy of suspected phaeochromocytoma lesions is contraindicated.

4.Malignant phaeos. The only reliable indicator of malignancy in phaeos is the presence of distant metastases or local invasion on histology. The histological appearance of the tumour cells themselves surprisingly has no significance.

5.Genetic testing. Up to 30% of phaeos, especially if familial, will have a genetic basis (see Table 2.9). Genetic testing is now recommended to be considered in all confirmed cases, especially in familial, syndromic, or recurrent cases. Fourteen gene loci have been associated with phaeochromocytoma—the commonest are shown in Table 2.9. Genetic testing can be targeted to limited loci according to the clinical presentation, the location of the tumour, and whether it is metastatic or not ( Further reading, p. 156).

OHCM 10e, pp. 228–9, p. 738, p. 837.

Table 2.9 Phaeochromocytomas: genetic mutations associated with phaeochromocytomas or paragangliomas

Gene	Syndrome	Associated features	Frequency
RET	MEN-2	Medullary carcinoma of the thyroid, hyperparathyroidism	5%
VHL	von Hippel–Lindau syndrome	Haemangioblastomata, renal, pancreatic tumours	8%
SDH B		Large, malignant, extra-adrenal paragangliomas	6%
SDH D		Often head and neck paragangliomas	4%
SDH C		Rare	0%
Neurofibromatosis	von Recklinghausen’s/disease	Skin neurofibromata, café-au-lait spots	4%

SDH, succinate dehydrogenase.

Further reading

Lenders JW, Duh QY, Eisenhofer G, et al.; Endocrine Society. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2014; 99: 1915–42.

Hypokalaemia

Persistent hypokalaemia (<2.5mmol/L) can cause muscle weakness, cramps, tetany, and polyuria, and exacerbate digoxin toxicity and predispose to cardiac arrhythmias. The majority of cases are due to the common causes (see Box 2.6) and are relatively easy to diagnose. However, puzzling cases where none of these features are present occur and prompt further investigation. A flow chart is shown in Fig. 2.8.

Box 2.6 Common causes of hypokalaemia *

•Diuretics

•Vomiting/diarrhoea

•Intestinal fistula

•Laxative or diuretic abuse

•Steroids (including fludrocortisone), liquorice, ACTH therapy

* See text for investigation of rare causes.

Note in Fig. 2.8 the importance of identifying the presence of acidosis and hypertension. Occult diuretic and purgative use should always be borne in mind. The commonest cause of persistent hypokalaemia with no other cause presenting in adulthood is Gitelman’s syndrome (NCCT-Na-Cl cotransporter defect), an asymptomatic congenital disorder, which can usually be separated from the rare, more severe Bartter’s syndrome (which usually presents neonatally or in early childhood and represents gene defects in the renal tubular proteins NKCC2, ROMK, or CLCNKB) by low serum Mg²⁺ levels.

OHCM 10e, p. 98, p. 674.

Fig. 2.8 Investigation of hypokalaemia.

Further reading

Shaer AJ. Inherited primary tubular hypokalemic alkalosis: a review of Gitelman and Bartter syndromes. Am J Med Sci 2001; 322: 316–22.

Hyperkalaemia

Artefactual and common causes need to be excluded, of which renal failure is the most important (see Table 2.10). If these fail to reveal a cause, then hypoadrenalism (which can be life-threatening), isolated mineralocorticoid deficiency, and type IV renal tubular acidosis (RTA) need to be excluded.

Table 2.10 Causes of hyperkalaemia

Artefactual	Other	Rare, but important
Sample left unseparated overnight	Excess K⁺ replacement	Hypoadrenalism
Sample haemolysed	K⁺-sparing diuretics, ACE inhibitors	Type IV RTA
Myeloproliferative disease (leakage of K⁺ from high cell counts)	Renal impairment, especially acute and after trauma or surgery Metabolic acidosis (especially DKA), rhabdomyolysis, burns, massive blood transfusion	Isolated mineralocorticoid deficiency

Hypoadrenalism is suggested by concomitant hyponatraemia, hypotension (including postural), malaise, and skin pigmentation. Diagnosis is by short Synacthen^® testing ( Adrenal failure, pp. 162–164). Note that hyperkalaemia is not a feature of 2° (pituitary) hypoadrenalism since aldosterone production is maintained by the renin–angiotensin system. Type IV RTA is common in patients with diabetes. It is associated with renal tubular dysfunction, as well as mildly impaired glomerular function. Serum creatinine is usually at, or above, the upper limit of normal. It is a state of hyporeninaemic hypoaldosteronism. Renin/aldosterone testing is suggestive, but there is no definitive test. Isolated mineralocorticoid deficiency is usually congenital (e.g. due to aldosterone synthase deficiency) but can be acquired (e.g. HIV disease). High renin and low aldosterone levels would be expected. Aldosterone resistance (pseudohypoaldosteronism with high aldosterone levels, but biochemical mineralocorticoid deficiency) has been described.

OHCM 10e, p. 93, p. 301, p. 674.

Adrenal failure

For causes of 1° adrenal failure, see Table 2.11.

Hypoadrenalism is often insidious in clinical onset. However, it is an important diagnosis to make, as it can be life-threatening, especially at times of stress. The key is to have a high index of suspicion. 1° adrenal failure is suggested by hyperkalaemia, hyponatraemia, hypotension (including postural), malaise, weight loss, nausea, abdominal pain, and skin pigmentation. In pituitary (2° adrenal failure, hyperkalaemia, hypotension, and pigmentation are absent, and malaise may be the only feature. Signs/symptoms of gonadal failure (e.g. loss of libido, reduced shaving, or amenorrhoea), if present, mandate exclusion of pituitary failure. Random cortisol levels can be misleading, as they may be high in the morning and low in the evening. Nonetheless, a random cortisol level >550nmol/L excludes the diagnosis and is a useful test in patients undergoing severe stress/illness (e.g. in intensive therapy unit (ITU)).

►Do not delay treatment. Where there is a strong suspicion of adrenal failure, treatment must not be delayed pending investigation. A short Synacthen^® test or random cortisol should be performed immediately and treatment commenced with steroids, awaiting results. Alternatively, treatment with dexamethasone 0.5mg daily (which does not cross-react in the cortisol assay) can be used and then discontinued for the day of testing. Patients on other forms of glucocorticoid therapy should discontinue treatment on the morning of the test and ideally 24h beforehand (12h for hydrocortisone or cortisone acetate). Mineralocorticoid replacement need not be discontinued.

Short ACTH (Synacthen^®) test

The standard test for adrenal failure is the short ACTH test. A low dose of synthetic ACTH (0.5 or 1.0µg) test was previously in vogue but has not been confirmed to be useful.

For 2° (pituitary) adrenal failure, alternative tests include the insulin tolerance test ( Insulin tolerance test, pp. 216–217) and the metyrapone test. However, these tests involve applying a stress and carry a risk in patients who are profoundly hypoadrenal. They are only indicated in patients within 6 weeks of pituitary surgery or with a pituitary insult where hypotrophy of the adrenal cortices has yet to develop.

Test to distinguish primary vs secondary adrenal failure

In the context of known pituitary disease and with failure of other pituitary hormones, adrenal failure can be assumed to be 2° (pituitary) in origin. Where isolated adrenal failure is identified, 1° adrenal failure is most likely and suggested by ↑ skin pigmentation and hyperkalaemia.

Table 2.11 Causes of 1° adrenal failure

Cause	Associated features	Diagnostic tests/notes
Autoimmune adrenalitis (>90% of cases in developed countries)	Autoimmune damage may be associated with polyglandular failure types 1 and 2	Anti-adrenal (21-hydroxylase) antibodies
Drugs	Ketoconazole, mitotane, etomidate, rifampicin, phenytoin	Exacerbate pre-existing adrenal impairment
TB	Extra-adrenal TB	Calcified or enlarged adrenals, extra-adrenal TB, but may only show shrunken glands
Other infections, e.g. histoplasmosis, syphilis	Seen in North and South America	Adrenal glands enlarged
Metastatic malignancy	Common with breast, lung, melanoma, or GI cancer, although does not always cause adrenal failure	Enlargement/deposits in adrenal glands on CT
Bilateral adrenal haemorrhage	Anticoagulation, adrenal vein sampling	Signs of haemorrhage on CT
AIDS	CMV/TB, Cryptococcus adrenalitis
Adrenoleukodystrophy	Especially in ♂ <15 years, dementia, quadriplegia
Adrenomyeloneuropathy	Neuropathy, blindness—may appear after adrenal failure
Familial glucocorticoid deficiency	Defective melanocortin 2 receptors, including Allgrove’s syndrome, hypoadrenalism associated with seizures, achalasia, and alacrima from childhood
Defective cholesterol metabolism
Congenital adrenal hypoplasia	Mutation in DAX1 or related genes, causing failure of adrenals to develop. Adrenal insufficiency from birth

Three additional tests can be used to confirm the level of adrenal failure

1.Basal plasma ACTH. This is usually the only additional test required. High levels are seen in 1° adrenal failure; ‘normal’ or low levels are seen in 2° adrenal insufficiency. Note that the sample must be taken and separated immediately at least 24h after the last dose of a short-acting glucocorticoid (e.g. hydrocortisone) to avoid pharmacological suppression. Patients on a longer-acting steroid may have to have the test repeated >24h after cessation of the steroid if the result is equivocal.

2.Anti-adrenal antibodies (anti-21 hydroxylase antibodies). These antibodies are present in around 70% of patients with autoimmune adrenalitis (Addison’s disease), the commonest cause of 1° adrenal insufficiency. However, they can also be present without adrenal failure in patients with other autoimmune conditions.

3.Long (depot) ACTH test. Chronic stimulation with ACTH can recover function in adrenal glands that have failed because of lack of pituitary ACTH, but not in 1° adrenal failure. This is given in the form of ACTH in oil on 2 consecutive days ( Long (depot) ACTH test, p. 225) or as an infusion over 48h. With the advent of reliable ACTH assays, this test is rarely indicated.

Additional diagnostic tests—exclude adrenoleukodystrophy in males

While the majority of cases of 1° hypoadrenalism are due to autoimmune disease in developed countries, there are multiple other rare causes. These should particularly be considered where adrenal failure occurs in childhood and/or is associated with neurological disease or hypogonadism (see Table 2.11). In particular, adrenoleukodystrophy (ALD) should be excluded. All ♂ diagnosed with 1° adrenal failure should have serum sent for very long-chain fatty acids (VLCFAs—raised in ALD), as early bone marrow transplantation (and, to a limited extent, treatment with ‘Lorenzo’s oil’) may prevent irreversible progressive neurological disease from developing (e.g. spastic paraparesis).

Further reading

Bornstein SR, Allolio B, Arlt W, et al. Diagnosis and treatment of primary adrenal insufficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2016; 101: 364–89.

Amenorrhoea

Amenorrhoea is often separated into 1° (never menstruated) and 2° (cessation of periods after menarche) amenorrhoea, but many causes are shared between the two categories. Structural assessment of the genital tract should be performed earlier in investigation of 1° amenorrhoea. Investigation of oligomenorrhoea is similar to 2° amenorrhoea. Menorrhagia and intermenstrual bleeding are due to different causes, often gynaecological in origin. ‘Irregular periods’ can fall into either category, depending on whether it actually refers to intermenstrual bleeding or variably spaced (anovulatory) periods. A plan of investigation is shown in Fig. 2.9.

In 2° amenorrhoea, it is helpful early on to identify 1° ovarian failure (e.g. due to Turner’s syndrome, premature ovarian failure, radiation, mumps orchitis, radiation, chemotherapy, or non-45XO gonadal dysgenesis) characterized by high gonadotrophins (LH, FSH). Where the gonadotrophins are equivocal or low, amenorrhoea due to hyperprolactinaemia or thyrotoxicosis should be excluded, but the commonest diagnosis is chronic anovulation due to polycystic ovarian syndrome. In this condition, the ovaries still produce oestrogen, resulting in a +ve progesterone withdrawal test; 10mg of medroxyprogesterone is given daily for 5 days and the test is +ve if any menstrual bleeding occurs in the following week. If the test is −ve, a pituitary (e.g. pituitary tumour) or hypothalamic (e.g. stress, anorexia nervosa, systemic illness, or weight loss) cause resulting in profound oestrogen deficiency must be considered.

Fig. 2.9 Investigation of amenorrhoea: (a) 1° and (b) 2°. See Delayed puberty, p. 176 for investigation of delayed puberty.

Further reading

Azziz R. The evaluation and management of hirsutism. Obstet Gynecol 2003; 101: 995–1007.

Infertility

Detailed assessment of infertility is beyond the scope of this text and is best referred to a specialist in this area. However, the general physician can take the following basic steps, always remembering that the couple should be assessed together as the problem may lie with the man, the woman, or a combination of both:

1.Semen analysis of the ♂ and, where possible, a post-coital test to confirm that live semen are delivered to the vaginal tract.

2.If amenorrhoea is present in the ♀, investigate as in Fig. 2.9.

3.If the ♀ is menstruating, determine if the cycles are ovulatory, e.g. by day 21, progesterone levels, or home measurement urinary dipstick of the LH surge.

If live semen are delivered and ovulation is occurring, then structural damage or chlamydial infection in the female genital tract is likely, and will require gynaecological assessment.

Hirsutism/virilization (raised testosterone)

Hirsutism refers to an ↑ in androgen-dependent terminal hairs in the ♀, typically over the face/chin, lower abdomen, arms and legs, and around the areola of the breast. Virilization reflects much higher androgen levels and comprises the features shown in Box 2.7. Over 20% of women have more androgen-dependent hair than they consider to be normal. In >95% of cases, this is associated with androgen levels in the ♀ normal range or slightly elevated in association with polycystic ovarian syndrome. Some drugs, such as ciclosporin, diazoxide, minoxidil, and androgenic steroids can also cause hirsutism. A history of recent onset (<6 months) and rapidly progressive hirsutism, particularly when associated with features of virilization and a testosterone level of >5nmol/L, should prompt a search for alternative adrenal or ovarian causes (see Fig. 2.10).

Box 2.7 Features of female virilization

•Clitoral enlargement

•Temporal hair loss

•Breast atrophy

•Deepening of voice

OHCM 10e, p. 230.

Fig. 2.10 Investigation of hirsutism. * CAH, congenital adrenal hyperplasia.

Further reading

Martin KA, Chang RJ, Ehrmann DA, et al. Evaluation and treatment of hirsutism in premenopausal women: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2008; 93: 1105–20.

Speiser PW, Azziz R, Baskin LS, et al.; Endocrine Society. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2010; 95: 4133–60.

Galactorrhoea (hyperprolactinaemia)

Galactorrhoea is always due to PRL and should be confirmed by asking the woman to demonstrate a secretion of milky appearance from the nipple. Rarely, galactorrhoea can occur with PRL levels in the normal range and regular menses, but usually it is associated with mildly raised levels and amenorrhoea in ♀ or very elevated levels in ♂. There is no link with breast size—gynaecomastia in ♂ is associated with excess oestrogen. Once dopamine-blocking drugs (major tranquillizers and anti-emetics, but not antidepressants), depot progesterone administration, and hypothyroidism have been excluded, all patients should have pituitary imaging to exclude a large tumour pressing on the pituitary stalk, especially if there are modest PRL levels (<10,000IU/L) associated with a large tumour (>2cm) (see Fig. 2.11). If the PRL levels are disproportionately low for the tumour size, serial dilution of the sample and re-estimation should be considered to exclude an artefactually low level due to the ‘hook effect’. Very high PRL levels (>10,000IU/L) are invariably associated with true prolactinomas. Nipple manipulation (e.g. to check if galactorrhoea has ceased) and chest wall trauma (including shingles) can also stimulate PRL levels.

Asymptomatic raised prolactin (macroprolactin)

If PRL is found (accidentally) to be persistently raised (>1000IU/L), but menstruation is normal and there is no galactorrhoea, consider the possibility of macroprolactin. This is a circulating complex of PRL multimers, and sometimes PRL autoantibodies of no biological importance, but gives a high reading in the PRL assay and the result often varies widely between assays. If the laboratory is alerted to a mismatch between PRL levels and the clinical picture, they can easily screen for this with polyethylene glycol precipitation. Stress and epileptic fits can result in transiently raised PRL levels, insufficient to cause galactorrhoea.

OHCM 10e, p. 236.

Fig. 2.11 Investigation of galactorrhoea.

Further reading

McKenna TJ. Should macroprolactin be measured in all hyperprolactinaemic sera? Clin Endocrinol (Oxf) 2009; 71: 466–9.

Melmed S, Casanueva FF, Hoffman AR, et al.; Endocrine Society. Diagnosis and treatment of hyperprolactinemia: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011; 96: 273–88.

Impotence/loss of libido/male hypogonadism

Symptoms and signs of hypogonadism in men (low testosterone levels)

•Reduced shaving.

•Loss of libido.

•Impotence.

•Reduced energy/aggression levels.

•Loss of pubic, chest, and axillary hair.

•Gynaecomastia often results due to a lower testosterone/oestrogen ratio.

Note that very low levels of testosterone (at least <5nmol/L, typical normal range 10–30nmol/L) are required to result in symptoms. Mild reductions are common, especially in the elderly, and are rarely of importance. Impotence alone (without loss of libido) can also be caused by neurovascular and psychological causes (e.g. diabetes, spinal damage, urological surgery, atherosclerosis of the aorta, drugs, stress, and psychosexual dysfunction). Where the testosterone level is at the lower limit of normal or SHBG abnormalities are suspected and symptoms are present, measurement of free testosterone or bioavailable testosterone by an established method may be indicated. Such methods may need referral to a reference laboratory.

After history taking for conditions described, investigation of suspected male hypogonadism requires

Hyperprolactinaemia or thyrotoxicosis, if present, need to be treated on their own merits. If the testosterone level is clearly low, high gonadotrophins point to testicular failure (e.g. testicular surgery, irradiation or trauma, chemotherapy, crypto-orchidism, previous orchitis, gonadal dysgenesis, including Klinefelter’s syndrome XXY). Low gonadotrophin levels with a clearly low testosterone level point to a hypothalamic or pituitary cause (systemic illness, pituitary tumour), which requires further investigations ( Hypopituitarism, pp. 128–130). If no cause is found for hypogonadotrophic hypogonadism, the likely cause is Kallman’s syndrome, especially if associated with anosmia.

OHCM 10e, p. 230.

Further reading

Bhasin S, Cunningham GR, Hayes FJ, et al.; Task Force, Endocrine Society. (2010) Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2010; 95: 2536–59.

Sato N, Katsumata N, Kagami M, et al. Clinical assessment and mutation analysis of Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18 sporadic patients. J Clin Endocrinol Metab 2004; 89: 1079–88.

Gynaecomastia

Gynaecomastia results from an excessive effect of oestrogens or a raised oestrogen/testosterone ratio. Causes are summarized in Table 2.12. True gynaecomastia should be associated with palpable breast tissue and distinguished from apparent breast enlargement due to obesity. Though very rare, the most important diagnoses to exclude are hypogonadism and testicular and lung tumours.

Table 2.12 Causes of gynaecomastia

Physiological	Newborn, adolescent, elderly
Hypogonadism	e.g. Klinefelter’s syndrome, testicular failure
↑ oestrogen	Testicular tumours, lung cancer producing hCG, liver disease, thyrotoxicosis
Drugs	Including oestrogens, spironolactone, cimetidine, digoxin, testosterone administration

Investigations should include

•Further review of drug history.

Physiological gynaecomastia should only be diagnosed if other causes have been excluded.

OHCM 10e, p. 230.

Delayed puberty

Definition

Puberty is considered delayed in girls if there is no breast development by age 13 (or menses by age 15) and in boys if there is no testicular enlargement by age 14. Note that 3% of normal children will fall into these categories.

Clinical features and initial investigations

A detailed history and examination is required for overt systemic disease, psychosocial stress, and anorexia nervosa, and to assess the child’s height, pubertal features (pubic hair, testicular size, breast growth, menses), and any dysmorphic features (e.g. features of Turner’s syndrome). Where possible, growth rate should be calculated from sequential height measurements over at least 6 months.

If no obvious cause is identified, baseline investigations should include:

•LH and FSH.

•TSH, FT4, PRL.

•FBC, U&E, bicarbonate (HCO₃⁻), CRP, and antigliadin/endomysial antibodies for occult systemic disease.

•Bone age.

This should enable the child to be placed in one of five categories:

1.Raised LH/FSH (1° gonadal failure). Causes: Turner’s syndrome, Klinefelter’s syndrome, ovarian/testicular injury. Proceed to karyotyping (should be performed in all girls with delayed puberty as Turner’s syndrome may not be apparent).

2.Short, low LH/FSH, overt systemic disease. Causes: asthma, anorexia nervosa, social deprivation, generalized illness, treatment for cancer, including cranial irradiation, dysmorphic (Noonan’s syndrome and others).

3.Short, low LH/FSH, occult systemic disease. Causes: hypothyroidism, hyperprolactinaemia, renal failure, RTA, coeliac disease, Crohn’s disease.

4.Short, low LH/FSH, no systemic disease. Causes: constitutional delay of puberty, hypothalamic/pituitary disease.

5.Not short, low LH/FSH. Causes: Kallman’s syndrome (if anosmia present) or isolated gonadotrophin deficiency. Cannot reliably distinguish from constitutional delay of puberty. Observe.

The investigation of children who fall into the commonest category ‘short, low LH/FSH, no systemic disease’ is summarized in Fig. 2.12. The onset of puberty after a period of observation is reassuring, but continued observation is required to ensure the process proceeds to completion, including a growth spurt. If not, further investigation for disorders of steroidogenesis, androgen insensitivity, skeletal dysplasia, premature gonadal failure, and, in the ♀, genital tract abnormalities and polycystic ovarian syndrome are indicated.

Fig. 2.12 Investigation of delayed puberty in children who are short, with no evidence of systemic disease¹ and low LH/FSH levels.

Notes:

1. Including normal thyroid function and PRL.

2. If develops headache, vomiting, or visual symptoms, proceed immediately to MRI.

3. Refer to a paediatric endocrinologist. Tests used vary, e.g. gonadotrophin response to luteinizing hormone-releasing hormone (LHRH) after androgenic priming and ITT for GH response.

Short stature

Evaluation of children who are below the third growth centile for age or particularly small for their family should include:

•Height for age (percentile).

•Mid-parental height (for girls, mean of father’s height minus 12.6cm + mother’s; for boys, add 12.6cm to mother’s height).

•Bone age (to assess growth potential/height prediction).

•Observation over 3–6 months to determine growth velocity.

Children of short (but normal) parents, who are growing normally, can be observed. Dysmorphic children require further evaluation/specialist assessment. Children who are short for their parental heights (low predicted height), particularly if growing slowly, and short children of pubertal age who have not entered puberty should be investigated as for ‘delayed puberty’. Referral for paediatric endocrinological assessment is advised.

Precocious puberty

Definition

Puberty is considered premature if multiple signs, including accelerated growth rate and bone age, appear by age 8 in girls or age 9 in boys. Note that isolated breast development (premature thelarche) or pubic hair (premature adrenarche) are benign conditions if no other evidence of puberty appears. True precocious puberty requires urgent investigation to determine the cause and avoid irreparable loss of final adult height. In girls, it is often idiopathic, but not in boys. The causes are given in Table 2.13.

Table 2.13 Causes of precocious puberty

Central	Gonadotrophin-independent
Idiopathic (especially girls)	Congenital adrenal hyperplasia (♂)
CNS hamartoma (especially pinealoma)	Adrenal/ovarian/hCG-secreting tumour
Other CNS diseases, e.g. hydrocephalus, trauma	McCune–Albright syndrome, hypothyroidism, follicular cyst (♀) familial testotoxicosis (♂)

Investigations

Precocious puberty is confirmed by pubertal levels of sex steroids (oestradiol, testosterone). Testicular enlargement (or ovarian enlargement on USS) and detectable LH/FSH levels suggest central precocious puberty and a CT/MRI scan of the brain is indicated. Gonadal enlargement can also be seen with testotoxicosis, hCG-producing tumours, hypothyroidism, and McCune–Albright syndrome. Further investigation should be performed in combination with a paediatric endocrinologist.

Thyroid function testing: general

In the majority of cases, thyroid function testing and interpretation are straightforward (see Fig. 2.13). However, the following points should be borne in mind.

1.Which first-line test?—TSH. TSH levels are the most sensitive indicator of thyroid dysfunction, except in patients with pituitary disease where they are uninterpretable. TSH used alone as a first-line test will miss (levels ‘normal’) unsuspected cases of 2° hypothyroidism, and some laboratories combine TSH and T4 as first-line tests. TSH is also unreliable in patients with recently treated hyperthyroidism, as it can remain suppressed (undetectable) for several weeks after FT4 or FT3 levels have normalized.

2.Which tests?—T4/T3. Free T3 and T4 tests (FT3, FT4) are now more reliable and preferred (although more expensive) to total T3 or T4 measurements. Interference in these assays does occur but is increasingly rare. Total thyroid hormone levels are markedly influenced by changes in binding proteins (e.g. due to pregnancy, oestrogen-containing contraceptives).

3.Thyroid autoantibodies. These are markers of autoimmune thyroid disease. Antithyroid microsomal antibodies have been identified as antithyroid peroxidase (anti-TPO) antibodies. Anti-TPO antibodies are more sensitive than anti-thyroglobulin (Tg) antibodies and are present in around 45–80% of Graves’ disease and 80–95% of Hashimoto’s disease/atrophic thyroiditis. Increasingly, laboratories are measuring anti-TPO directly as their only antibody test (sometimes just referred to as ‘antithyroid antibodies’). Note that anti-TSH receptor antibodies—the cause of Graves’ disease—require a specific assay request. (For indications for testing, Anti-TSH receptor antibody testing, p. 184).

4.Tests should agree. To confirm thyroid dysfunction, at least two TFTs and, in cases of doubt, all three (TSH, FT3, FT4) should be performed. Results of the tests should be in agreement—if not, assay interference (heterophile antibodies, anti-T4 or anti-T3 antibodies present in the serum) or unusual causes should be suspected.

5.Avoid thyroid function testing in systemically unwell patients. In very ill patients, especially in intensive care, a pattern of ‘sick euthyroidism’ is often seen, with low TSH levels, low FT3 levels, and sometimes low FT4 levels. Accurate interpretation of true thyroid status is impossible. A raised FT3 level in a very ill patient suggests significant hyperthyroidism, and a very raised TSH level (>20mU/L) with undetectable FT4 levels suggests profound hypothyroidism. Other changes should be interpreted with extreme caution and the tests repeated after recovery.

OHCM 10e, pp. 216–17.

Fig. 2.13 Patterns of TFTs. To use this table, you need the results of both TSH and either free T4 (FT4) or free T3 (FT3) tests. If either FT4 or FT3 are outside the reference range, then FT4/FT3 are considered abnormal in this table. If FT4 and FT3 are abnormal in different directions (e.g. one is low and the other is high), see point 4, ‘Tests should agree’, pp. 180–181.

Reprinted from The Lancet, 357, Dayan CM ‘Interpretation of thyroid function tests’ 619–24 (2001) with permission from Elsevier.

Further reading

Association for Clinical Biochemistry/British Thyroid Association (2006). UK guidelines for the use of thyroid function tests. London: ACB/BTA. http://www.acb.org.uk/docs/default-source/guidelines/TFTguidelinefinal.pdf.

National Academy of Clinical Biochemistry Practice Guidelines (2002). http://www.aacc.org/members/nacb/LMPG/Pages/default.aspx.

Hyperthyroidism (thyrotoxicosis)

Clinical features

Hyperthyroidism is rare in childhood but affects all adult age groups. Classic features include weight loss despite ↑ appetite, palpitations, AF, heat intolerance, anxiety, agitation, diarrhoea, tremor, and proximal weakness. Lid retraction and lid lag can be seen in any cause of hyperthyroidism, but proptosis, periorbital oedema, chemosis, diplopia, and optic nerve compression only occur in association with Graves’ disease (thyroid eye disease) and occasionally associated with pretibial myxoedema and thyroid acropachy. In the elderly, presentation with isolated weight loss or AF is common. Raised ALP and SHBG, leucopenia, and rarely hypercalcaemia are recognized associations.

Thyroid function testing

An undetectable TSH level and an ↑ free T3 level are required to diagnose hyperthyroidism. In milder cases, T4 levels may be in the normal range (‘T3 toxicosis’). Normal TSH levels with ↑ T4 and T3 are seen in TSH-secreting pituitary tumours (very rare) or in patients with thyroid hormone resistance (also very rare) (see Fig. 2.13).

Investigation of cause

For an overview of investigation, see Fig. 2.14.

Under the age of 40, Graves’ disease is the commonest cause. After this age, Graves’ disease, toxic nodular goitre, and toxic nodule all occur. However, a short history (1 month) of symptoms or absence of relevant symptoms (chance blood test finding) raises the possibility of self-resolving (transient) thyroiditis, a diagnosis supported by neck pain and raised ESR (viral/subacute/De Quervain’s) or occurrence in the first 9 months postpartum (postpartum thyroiditis—painless). Transient thyrotoxicosis can also occur in patients with subclinical autoimmune thyroiditis (‘silent thyroiditis’—painless), especially during cytokine therapy (e.g. interferon for hepatitis C). Graves’ disease or other forms of autoimmune thyroiditis are seen in >30% of patients 2–3 years after treatment with alemtuzumab (for MS), in association with lymphocyte repopulation. If self-resolving thyroiditis is suspected, withhold treatment and repeat the tests after 6 weeks. When thyroid eye disease is present, no further tests are required to diagnose Graves’ disease. If not, antithyroid antibodies (e.g. anti-TPO antibodies) and isotope thyroid scanning can be useful to distinguish possible causes (see Fig. 2.14 and Chapter 14). No uptake is seen in transient thyroiditis. Excess thyroid hormone ingestion rarely causes very marked thyrotoxicosis, unless the active form (T3) is taken (T3 tablets or desiccated thyroid extract).

Iodine

Iodine has multiple and conflicting effects on the thyroid. Potassium iodide inhibits release of thyroid hormones from the gland and thyroid hormone biosynthesis (Wolff–Chaikoff effect), promoting hypothyroidism. However, escape from these effects occurs in most individuals in a few weeks. In patients with a multinodular goitre, excess iodine (e.g. in amiodarone or radiographic contrast media) can result in thyrotoxicosis by excess provision of substrate (Jod–Basedow effect).

Amiodarone

Has three main effects on the thyroid hormone axis:

•Inhibits T4 → T3 conversion, which in the pituitary can result in an asymptomatic mild rise in TSH level (reduced thyroid hormone action) and/or a rise in FT4 level.

•Can induce true hypothyroidism, usually in the first year of treatment.

•Can induce true hyperthyroidism either via the Jod–Basedow effect in patients with multinodular goitre or by destructive thyroiditis in healthy glands.

Thyrotoxicosis can occur at any time after commencing therapy and can be very difficult to treat. Interpretation of TFTs on amiodarone: a raised FT3 level indicates true hyperthyroidism; a markedly raised TSH level (e.g. >10mU/L), especially if the FT4 level is low, indicates true hypothyroidism.

Fig. 2.14 Investigation of the cause of hyperthyroidism.

Hyperthyroidism in pregnancy

Significant hyperthyroidism in pregnancy is generally due to Graves’ disease. Mild hyperthyroidism, particularly in association with hyperemesis gravidarum in the first trimester, is often due to a cross-reaction by very high hCG levels with the TSH receptor (‘gestational thyrotoxicosis’). In the postpartum period, thyrotoxicosis may be due to postpartum thyroiditis (self-resolving) or a recurrence of Graves’ disease (requires treatment). Measurement of anti-TSH receptor antibody levels may be indicated to distinguish these possibilities.

Thyroid storm

This is defined as severe thyrotoxicosis with confusion/delirium not explained by other factors. There is no definitive test and levels of thyroid hormone are not higher than in other thyrotoxic individuals with no features of storm. Severe agitation, tachycardia, and hyperpyrexia are usually seen. Usually precipitated by infection, trauma, or surgery, especially to the thyroid gland. Very rare but tends to occur in individuals who have been poorly compliant in the first few weeks of drug therapy for thyrotoxicosis.

Anti-TSH receptor antibody testing (TBII, TRAbs. TSAb)

This test is increasingly available in local and regional laboratories. In second- or third-generation assays, especially assays for stimulatory antibodies, it is positive in >95% of cases, save other tests, and indicated that the patient is at risk of Graves’ eye disease. Indications for anti-TSH receptor antibody testing include distinguishing gestational thyrotoxicosis or postpartum thyroiditis from Graves’ disease, indicating the risk of neonatal thyrotoxicosis and (controversial) predicting recurrence after a course of thioamide drug therapy.

OHCM 10e, p. 31, p. 216, p. 562.

Further reading

Barbesino G, Tomer Y. Clinical utility of TSH receptor antibodies. J Clin Endocrinol Metab 2013; 98: 2247–55.

Hypothyroidism

Clinical features

Classic clinical features of hypothyroidism include weight gain, cold intolerance, dry skin, constipation, memory loss, lethargy/slow thought/‘slowing up’, menorrhagia, periorbital/facial oedema, loss of the outer two-thirds of eyebrows, deafness, chest pain, and coma. These are rarely seen nowadays, as TFTs are easy to perform and detect the disease usually at an earlier stage. Weight gain, dry skin, and lethargy are frequently reported, but even biochemically hypothyroid individuals can only confidently be ascribed to thyroid status if they reverse on treatment.

Biochemical diagnosis

↑ TSH with T4 in the normal range is referred to as subclinical hypothyroidism. ↑ TSH with ↓ T4 is overt hypothyroidism. ↓ T4 with TSH in the normal range may also be due to pituitary failure (2° hypothyroidism) and, if persistent, requires pituitary function testing. See Fig. 2.13 for other patterns of TFTs.

Differential diagnosis (causes)

In iodine-sufficient countries, most spontaneous hypothyroidism is due to autoimmune thyroiditis (Hashimoto’s disease if goitre present, atrophic thyroiditis if goitre absent)—antithyroid antibodies (anti-TPO, anti-Tg) present in 80–90% of cases. Other common causes are post-thyroidectomy, post-radioiodine therapy, and side effects of amiodarone or lithium. Rarer causes include treatment with cytokines or other drugs (e.g. interferons, granulocytic macrophage colony-stimulating factor (GM-CSF), interleukin-2, tyrosine kinase inhibitors, alemtuzumab), vast excess of iodine intake (iodine drops, water purifying tablets), congenital hypothyroidism (caused by a variety of genetic defects; should be detected by neonatal screening programme), iodine deficiency (urinary iodide excretion <45µg/day, commonest cause worldwide, especially mountainous areas, S. Germany, Greece, Paraguay—‘endemic goitre’), thyroid-blocking substances in the indigenous diet (goitrogens, especially in brassicas and cassava, e.g. in Sheffield, Spain, Bohemia, Kentucky, Virginia, Tasmania—‘endogenous goitre’ without iodine deficiency), Pendred’s syndrome (mild hypothyroidism with sensorineural deafness due to Mondini cochlear defect detectable on MRI, positive perchlorate discharge test). For further information, Transient hypothyroidism, p. 187.

►► Diagnostic catches ↑ TSH and ↓ T4 always represents hypothyroidism. If the TSH alone is ↑ and the T4 is not even slightly low, a heterophile antibody interfering in the TSH assay may be present in the patient’s serum. This is especially likely if there is no change in TSH level after thyroxine treatment, but the T4 level rises (confirming compliance with tablets). For unusual patterns of thyroid function tests, see Fig. 2.14. Note that, within the first 1–3 months (or longer) after treatment of hyperthyroidism, profound hypothyroidism may develop with a ↓ T4, but the TSH may still be suppressed or only mildly raised due to the long period of TSH suppression prior to treatment. Raised TSH alone with disproportionate symptoms of lethargy may be seen in hypoadrenalism.

Transient hypothyroidism

Transient/self-resolving hypothyroidism, often preceded by hyperthyroidism, is seen in viral thyroiditis, after pregnancy (postpartum thyroiditis), and in some individuals with autoimmune thyroiditis. Treatment temporarily with levothyroxine is only required if the patient is very symptomatic. Thyroid function should return to normal within 6 months.

Subclinical hypothyroidism

A raised TSH (<20mU/L) with normal T4/T3 is very common and seen in 5–10% of women and ~2% of ♂. It is usually due to subclinical autoimmune thyroid disease and is frequently discovered on routine testing. In randomized trials, ~20% of patients obtain psychological benefit from beginning T4 therapy; in many others, it is probably truly asymptomatic. If antithyroid antibodies are detectable, the rate of progression to overt hypothyroidism is ~50% at 20 years, but higher than this with higher initial TSH levels. If the TSH alone is raised with −ve antibodies (or the TSH is normal with raised antibodies alone), overt hypothyroidism develops in 25% at 20 years. A reasonable approach is a trial of levothyroxine for 6 months in symptomatic patients with subclinical hypothyroidism or TSH >10mU/L, and observing the TSH level at 6- to 12-monthly intervals in asymptomatic patients with TSH <10mU/L.

Hypothyroidism and pregnancy

Overt hypothyroidism is associated with poor obstetric outcomes. Recent evidence suggests that subclinical hypothyroidism is associated with an ↑ miscarriage rate and a slight reduction in the baby’s intelligence quotient (IQ) and should be treated. Some authorities advocate screening for hypothyroidism in all antenatal patients as early as possible in pregnancy. Patients on T4 need to ↑ their dose by 25–50µg from the first trimester of pregnancy. Maternal levothyroxine can compensate for fetal thyroid failure in utero, but congenital hypothyroidism must be detected at birth (screening test) to avoid mental retardation developing. Where the mother and fetus are both hypothyroid—most commonly due to iodine deficiency—mental retardation can develop in utero (cretinism). Note that mothers with +ve antithyroid antibodies and/or subclinical hypothyroidism have a 50% chance of developing (transient) postpartum thyroiditis.

OHCM 10e, p. 31, p. 149, p. 203.

Further reading

Cooper DS, Biondi B. Subclinical thyroid disease. Lancet 2012; 379: 1142–54.

Hypercalcaemia

Clinical features

Usually asymptomatic if Ca²⁺ <3.0mmol/L. Typical symptoms include polydipsia/polyuria, constipation, indigestion, pancreatitis, hypertension, tiredness, drowsiness/confusion, abdominal pains, and renal colic. Renal failure can occur due to dehydration (reversible), nephrocalcinosis, and/or staghorn calculi. Osteitis fibrosa cystica in cases of hyperparathyroidism (with subperiosteal resorption of bone, particularly of the distal phalanges and bone cysts—brown tumours) is now rare, other than in renal failure, but can be associated with bone pain.

Investigation of the cause

Ninety-five per cent of persistent hypercalcaemia is due to either hyperparathyroidism or malignancy (see Fig. 2.15). Asymptomatic 1° hyperparathyroidism is common in 50- to 70-year-old women, and a PTH level (sampled simultaneously with ↑ Ca²⁺ and measured in a highly sensitive assay) which is raised or in the upper normal range in the presence of hypercalcaemia confirms the diagnosis. Low normal or low levels of PTH should prompt a search for malignancy, especially breast, prostate, bronchus, kidney, thyroid, or myeloma. Bone-derived ALP levels may be raised in both malignancy and hyperparathyroidism. Bone scan may be useful in disseminated malignancy but can be −ve in cancers releasing PTH-related peptide (NOT detected in routine PTH assays) and in myeloma. If malignancy is not found, the conditions shown in Fig. 2.15 need to be considered. Markedly abnormal renal function is seen in milk-alkali syndrome, myeloma, and tertiary hyperparathyroidism. Sarcoid may be difficult to diagnose but is suggested by a raised serum ACE level (not invariable), a dramatic response to steroids, and a +ve liver or other biopsy for granulomata.

Investigation of established hyperparathyroidism

Familial benign hypocalciuric hypercalcaemia (FBHH) is a very rare condition caused by an inactivating mutation of the calcium-sensing receptor (CaSR). This results in stable, lifelong hypercalcaemia with a raised PTH level, which rarely causes complications. It is inherited in an autosomal dominant fashion.

The hallmark is hypocalciuria—defined as:

(All in units of mmol/L.)

Although rare, it is important to recognize, as parathyroidectomy is not required.

Further investigation of 1° hyperparathyroidism should include serum creatinine, KUB plain abdominal X-ray to exclude renal stones, and a spot urine Ca²⁺/creatinine to rule out FBHH.

In >80% of cases, 1° hyperparathyroidism is due to adenomatous change in one of the four parathyroid glands. In a minority of cases and in familial hyperparathyroidism associated with MEN-1 (pituitary tumours, endocrine pancreatic tumours, and hyperparathyroidism) or MEN-2 (medullary carcinoma of the thyroid, phaeochromocytoma, and hyperparathyroidism) four gland hyperplasia occurs, requiring resection of at least three-and-a-half glands for treatment. Very rarely (<1% of cases), parathyroid carcinoma is the cause. Lithium therapy may also be associated with (mild) hyperparathyroidism. Once a diagnosis of 1° hyperthyroidism is made, ^99mtechnetium-sestamibi radionucleotide scanning is the most sensitive imaging technique and will show the location of the parathyroid adenoma. In difficult cases, local venous sampling may be required for localizing a parathyroid adenoma, especially if it is outside the neck.

Tertiary hyperparathyroidism

Refers to acquired autonomy of the parathyroid glands leading to hypercalcaemia following chronic vitamin D deficiency, as seen in renal failure or with malabsorption. 2° hyperparathyroidism is associated with hypocalcaemia and is the appropriate response to vitamin D deficiency.

Fig. 2.15 Investigation of hypercalcaemia.

Hypocalcaemia/osteomalacia

Clinical features

Chronic hypocalcaemia is often surprisingly asymptomatic. Symptoms and signs, when present, include muscle spasms, paraesthesiae, especially around the mouth and in fingers, tetany, fits, +ve Chvostek’s (VIIth nerve hyperexcitability) and Trousseau’s signs (tetany of the hand when BP cuff inflated). Chronic hypocalcaemia is also associated with papilloedema, abnormal dentition (if begins in childhood), cataract, and intracranial calcification (of no clinical consequence). Hypocalcaemia due to vitamin D deficiency is associated with muscle pains, proximal myopathy, and osteomalacia. In some cases of pseudohypoparathyroidism (type Ia), there are phenotypic abnormalities (somatic features), including short fourth metacarpal, bone changes (Albright’s hereditary osteodystrophy), mental retardation, short stature, obesity, and resistance to other hormones, e.g. TSH, glucagon, gonadotrophins.

Investigation of cause

Persistent hypocalcaemia (corrected for serum albumin levels) with a normal serum creatinine level is almost always due to either hypoparathyroidism or vitamin D deficiency (osteomalacia). Other causes and distinguishing features are shown in Table 2.14, and a scheme for diagnosis is shown in Fig. 2.16. In failure of PTH action, Ca²⁺ is very low (<1.8mmol/L) and PO₃⁴⁻ is raised, but ALP is not raised and there is no osteomalacia. If the PTH is found to be raised, then pseudohypoparathyroidism can be diagnosed, which is subclassified as type Ia (paternally inherited a Gs-α defect with somatic features), type Ib (‘renally selective’ maternally inherited Gs-α defect—no somatic features), or type II. The classic test is the Elsworth–Howard test—measuring urine cyclic adenosine monophosphate (cAMP) response to infused PTH(1–34) analogue. Families with type 1a may also include patients with pseudopseudohypoparathyroidism, characterized by normocalcaemia, but somatic changes of pseudohypoparathyroidism. The mutations are in the same gene. The aetiology of type II pseudohypoparathyroidism (normal renal Ca²⁺ excretion and no other somatic features) remains unclear.

A low or normal PTH in the presence of a Ca²⁺ level <1.8mmol/L makes hypoparathyroidism the likely diagnosis (see Table 2.14 for possible causes), but an attempt to rule out an activating CaSR mutation with a urine Ca²⁺/creatinine ratio (see Fig. 2.15) should be made. In this rare genetic condition, Ca²⁺ levels are generally higher (around 1.75mmol/L). Importantly, Ca²⁺ or vitamin D replacement has a high likelihood of causing nephrocalcinosis and is best avoided. Autoimmune hypoparathyroidism in children or young people is particularly seen in association with autoimmune polyglandular syndrome type 1 (chronic candidiasis, coeliac disease, adrenal insufficiency).

In failure of vitamin D action (see Fig. 2.16, Table 2.14), there is a compensatory PTH rise, which partly corrects the Ca²⁺ level but causes a raised ALP. In addition, there is osteomalacia. In the presence of a significantly raised creatinine, renal osteodystrophy (impaired 25-OH vitamin D generation) is the most likely diagnosis; otherwise dietary vitamin D deficiency (low 25-OH vitamin D) or vitamin D resistance must be distinguished (see Table 2.14).

Osteomalacia

Osteomalacia is strictly a histological diagnosis but is suggested by Looser’s zones and pseudo-fractures on X-ray (especially pelvis and upper femur). If osteomalacia with muscle weakness occurs in the absence of hypocalcaemia or a raised ALP, hypophosphataemia (‘vitamin D-resistant rickets’) is likely. Causes of a low PO₃⁴⁻ include intrinsic renal disease, congenital PO₃⁴⁻ leak, acquired PO₃⁴⁻ leak, and oncogenic osteomalacia. This last condition is associated with very difficult-to-find tumours, often benign, typically haemangiopericytomas of the naso-/oropharynx that may take years to become manifest. It is due to secretion of the cytokine fibroblast growth factor 23 (FGF23) by tumours, causing a phosphaturic effect. If suspected, FGF23 levels can be assayed in specialized laboratories. Treatment is PO₃⁴⁻ replacement until the tumour can be resected.

Fig. 2.16 Investigation of hypocalcaemia.

Table 2.14 Causes of hypocalcaemia

Cause	Features
*Lack of PTH action*	*Very low Ca²⁺, high PO₄³⁻, normal ALP*
Hypoparathyroidism	Autoimmune, post-neck surgery (may be transient), radioiodine, congenital (e.g. di George)
Pseudohypoparathyroidism	Type Ia (paternally inherited Gs-α mutation plus somatic features) Type Ib (maternally inherited, no somatic features, ‘renally selective’) Type II (no somatic features, normal Ca²⁺ excretion
Hypomagnesaemia	Inhibits PTH release
Activating CaSR mutation	Indistinguishable from 1° hypoparathyroidism, except present from childhood and urinary Ca²⁺/creatinine ratio not low
*Failure of vitamin D action*	Ca²⁺*not very low (>1.8mmol/L),↑ALP,↓PO₄³⁻, ↑PTH, osteomalacia*
Vitamin D deficiency	Dietary/↓ sunlight, malabsorption, ↑ metabolism (phenytoin, rifampicin)
Renal failure	Failure of 1-hydroxylation of vitamin D
Inherited failure of 1-α hydroxylase (vitamin D-dependent rickets type I)	Normal 25-OH vitamin D, ↓ 1,25-OH vitamin D levels
Vitamin D receptor defect (vitamin D-dependent rickets type 2 *)	Normal 25-OH, normal 1,25-OH vitamin D levels
*Other*
Acute pancreatitis	Transient
Hungry bone syndrome—immediately post-parathyroidectomy	Transient
Drugs, e.g. foscarnet, bisphosphonates, ethylenediamine tetra-acetic acid (EDTA), citrate in blood	Transient
Neonatal (with prematurity)	Transient

* ‘Vitamin D-resistant rickets’ is rickets with normal Ca²⁺ and vitamin D due to X-linked hypophosphataemia.

Further reading

Ward BK, Magno AL, Walsh JP, Ratajczak T. The role of the calcium-sensing receptor in human disease. Clin Biochem 2012; 45: 943–53.

Diabetes mellitus

Diagnosing diabetes mellitus

Diabetes is defined as a state of chronic hyperglycaemia at levels that, if untreated, would result in microvascular complications (e.g. retinopathy). Adverse pregnancy outcomes (and ↑ macrovascular risk) occur at a lower level of glucose and hence lower cut-offs are used in pregnancy. However, since blood glucose levels vary through the day following meals, physical activity, and stress, defining these levels of glucose with a single test or a short dynamic test has proved difficult.

Figure 2.17 provides a scheme for testing for diabetes. Table 2.15 provides the American Diabetes Association (ADA) and WHO criteria for the diagnosis of diabetes, which differ slightly and have been updated since 2013. One of four different criteria can now be used to diagnose diabetes. Note that HbA_1c can now be used to diagnose diabetes if performed in a standardized Diabetes Control and Complications Trial (DCCT)-aligned assay and has the advantage that a fasting sample is not required. HbA_1c for diabetes diagnosis is not recommended using point-of-care machines (not standardized). In conditions where there is ↑ RBC turnover, such as pregnancy (second and third trimesters), recent blood loss or transfusion, Epo therapy, or haemolysis, HbA_1c should not be used for diagnosis. HbA_1c may also be problematic in the presence of haemoglobinopathies, unless specific assays not influenced by abnormal Hb are used. It should also be noted that the four different criteria for diagnosing diabetes do not completely overlap, e.g. a person may have fasting blood sugar of 6.8mmol/L (= impaired fasting glucose (IFG)/↑ diabetes risk) but an HbA_1c of 55mmol/mol (= diabetes). Hence only one test should be used.

Oral glucose tolerance testing is rarely required (and only recommended in pregnancy by the ADA) but can sometimes be useful to make the diagnosis of ↑ diabetes risk in borderline cases (see Table 2.15).

Fig. 2.17 Diagnosis of diabetes.

Table 2.15 ADA and WHO criteria for the diagnosis of diabetes

	ADA (2016)	WHO (2006, 2013)
Normoglycaemia	Fasting plasma glucose <5.6mmol/L (100mg/dL)*	Fasting plasma glucose <6.1mmol/L (110mg/dL)*
Normoglycaemia	OR 2h post-75g load of plasma glucose <7.8mmol/L (140mg/dL)	OR 2h post-75g load of plasma glucose <7.8mmol/L (140mg/dL)
Diabetes	Casual plasma glucose >11.1mmol/L (200mg/dL)¹	Casual plasma glucose >11.1mmol/L (200mg/dL)¹
	OR fasting plasma glucose >7.0mmol/L (126mg/dL)¹	OR fasting plasma glucose 7.0mmol/L (126mg/dL)¹
	OR 2h post-75g load of plasma glucose >11.1mmol/L (200mg/dL)²	OR 2h post-75g load of plasma glucose >11.1mmol/L (200mg/dL)
	OR HbA_1c >48mmol/mol (6.5%)	OR HbA_1c >48mmol/mol (6.5%)
IFG	Fasting plasma glucose 5.6–6.9mmol/L*	Fasting plasma glucose 6.1–7.0mmol/L (126mg/dL)
IFG		BUT not officially recognized—recommend progress to 2h 75g glucose tolerance test (GTT)*
Impaired glucose tolerance (IGT)	Not routinely measured 2h post-75g load of plasma glucose 7.8–11.1mmol/L (140–199mg/dL)	2h post-75g of glucose 7.8–11.1mmol/L (140–199mg/dL)
↑ risk of diabetes (pre-diabetes)—includes IFG and IGT also	HbA_1c: 5.7–6.4% (39–46mmol/mol)
Gestational diabetes	Casual plasma glucose >11.1mmol/L (200mg/dL)	Casual plasma glucose 11.1mmol/L
	OR fasting plasma glucose 7.0mmol/L (126mg/dL)*	OR fasting plasma glucose >7.0mmol/L
	OR two or more of the following plasma glucose values after 75g glucose load: fasting >5.3mmol/L; 1h >10mmol/L; 2h >8.6mmol/L (155mg/dL)*	OR one or more of the following: fasting 5.1–6.9mmol*; or following 75g glucose load: 1h post-glucose >10mmol/L; 2h post-glucose 8.5–11.0mmol/L

* ADA and WHO criteria differ.

¹ If the patient does not have classic symptoms (polyuria, polydipsia, unexplained weight loss), then this test should be repeated on a different day.

² Not recommended by the ADA for routine clinical use.

Notes

•If symptoms are not present, tests must be repeated on two occasions, ideally more than a week apart, to confirm that levels are indeed chronically raised.

•Values are given for venous plasma glucose. Capillary blood glucose are ~1.0mmol/L higher than venous plasma.

•If the patient has an intercurrent illness (e.g. infection or MI), tests should be repeated once the patient has recovered.

•Different criteria are used to diagnosis diabetes in pregnancy (gestational diabetes).

Blood samples for glucose testing: in unseparated whole blood, glycolysis by red cells reduces glucose levels by 10–15% per h at room temperature, leading to falsely low results. Clotted (serum) samples without preservative can be used for glucose measurements if the sample is separated rapidly; once separated, the glucose level is stable for 8h at room temperature and 72h at 4°C. Alternatively, a tube containing fluoride oxalate to inhibit glycolysis can be used if the sample is to be kept unseparated at room temperature for many hours.

False +ve diagnoses may arise if the subject has prepared inadequately (see Box 2.8).

Box 2.8 Preparation for a fasting blood test

•Refrain from any food or drink from midnight before the morning of the test.

•Water only is permitted.

•Regular medication can generally be deferred until a blood sample has been taken.

•The appropriate sample is taken between 8 a.m. and 9 a.m. the following morning.

This preparation is also required for a 75g oral glucose tolerance test (OGTT) or for measurement of fasting blood lipids. Fasting blood tests should be avoided in insulin-treated patients—risk of hypoglycaemia. Fasting is defined by the ADA as no caloric intake for at least 8h.

Normoglycaemia

The diagnostic criteria for normoglycaemia are given in Table 2.15 (note the difference between ADA and WHO for FPG cut-off). Note that there is NO defined random plasma glucose that confirms normoglycaemia, and this complicates the development of screening strategies for diabetes. However, if a random value is <5.6mmol/L, diabetes is unlikely.

Impaired glucose tolerance

This is the preferred diagnostic category for pre-diabetes used by the WHO. The diagnosis of IGT can only be made using a 75g OGTT; a random blood glucose measurement will often point to the diagnosis when other results are non-diagnostic.

This category denotes a stage intermediate between normal glucose levels and DM. By definition, plasma glucose levels are not raised to DM levels, so typical osmotic symptoms are absent. Although subjects with IGT are not at direct risk of developing chronic microvascular tissue complications, the incidence of macrovascular complications (i.e. coronary heart disease (CHD), cerebrovascular disease, peripheral arterial disease (PAD)) is ↑. Presentation with one of these conditions should therefore alert the clinician to the possibility of undiagnosed IGT (or type 2 DM). Note that up to 25% of individuals who are diagnosed with IGT by an OGTT may revert to normal on re-testing.

Impaired fasting glucose

This is the diagnostic category for pre-diabetes preferred by the ADA and depends on the FPG. Instructions for fasting blood test are shown in Box 2.8. The revised 2005 criteria lowered the lower limit for diagnosing IFG, so the range according to the ADA is 5.6–7.0mmol/L. This category is also usually asymptomatic. To date, cross-sectional studies suggest that IGT and IFG may not be synonymous in terms of pathophysiology and long-term implications, and a proportion of patients will fall into one category, but not the other. Also some patients with fasting blood glucose <7.0mmol/L may have 2h blood glucose on the OGTT of >11.1mmo/L and hence would have diabetes by the WHO criteria. If an OGTT is performed, the 2h value takes precedence over the fasting value in the diagnosis of diabetes if the values do not agree.

Oral glucose tolerance test

The OGTT (see Box 2.9) continues to be regarded as the most relevant means for establishing the diagnosis of diabetes in equivocal cases, although its reproducibility is poor. In borderline cases, the WHO suggests that only when an OGTT cannot be performed does the diagnosis rely on FPG. It is also the preferred approach in pregnancy. OGTTs should be carried out under controlled conditions after an overnight fast.

The interpretation of the 75g GTT is shown in Table 2.16. These results apply to venous plasma. Marked carbohydrate depletion can impair glucose tolerance; the subject should have received adequate nutrition in the days preceding the test.

Box 2.9 Oral glucose tolerance test

•Preparation: 3-day unrestricted CHO intake and activity. No medication on day of test. 8–14h fast. No smoking.

•75g ¹ of anhydrous glucose is dissolved in 250mL of water; flavouring with sugar-free lemon and chilling ↑ palatability and may reduce nausea. The subject sits quietly throughout the test.

•Blood glucose is sampled before (time 0), and at 120min after, ingestion of the drink, which should be completed within 5min.

•Urinalysis may also be performed every 30min, although it is only of interest if a significant alteration in renal threshold for glucose is suspected.

¹ In children, 1.75g/kg, up to 75g.

Effect of intercurrent illness on glycaemia

Patients under physical stress associated with surgery, trauma, acute MI, acute pulmonary oedema, or stroke may have transient ↑ of plasma glucose—often settles rapidly without antidiabetic therapy. However, the hormonal stress response in such clinical situations is liable to unmask pre-existing DM or to precipitate DM in predisposed individuals. Blood glucose should be carefully monitored and the urine tested for ketones. Sustained hyperglycaemia, particularly with ketonuria, demands vigorous treatment with insulin in an acutely ill patient. Re-testing is usually indicated following resolution of the acute illness—an OGTT at a 4- to 6-week interval is recommended if glucose levels are equivocal.

Table 2.16 Interpretation of the 75g OGTT (WHO)

	Venous plasma glucose (mmol/L)
	Fasting	120min post-glucose load
Normal	<6.0	<7.8
IFG	6.1–6.9	N/A
IGT	N/A	7.8–11.0
DM	>7.0	>11.1

1. In the absence of symptoms, a diagnosis of diabetes must be confirmed by a second diagnostic test on a separate day.

2. For capillary whole blood, the diagnostic cut-offs for diabetes are >6.1mmol/L (fasting) and 11.1mmol/L (120min). The range for IFG based on capillary whole blood is >5.6 and <6.1mmol/L. In the diagnosis of diabetes, the 2h post-blood value predominates if values do not agree.

Screening for diabetes

This remains controversial, and universal screening is not generally advocated, even though rates in the adult population exceed 5% in almost all countries and >10% in many countries. A low threshold for testing in those at high risk is advocated. The ADA suggests that 3-year screening in asymptomatic adults be considered over the age of 45, and especially where the BMI is >25 and there is high-risk ethnicity (including African or South Asian origin), a first-degree relative with diabetes, or women who have had a large baby (>9lb) or a previous diagnosis of gestational diabetes.

Further reading

American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2016; 39 (Suppl 1), S13–22.

World Health Organization 2006 Guidelines: http://www.who.int/diabetes/publications/Definition%20and%20diagnosis%20of%20diabetes_new.pdf

World Health Organization (2013). Diagnostic criteria and classification of hyperglycaemia first detected in pregnancy. Geneva: World Health Organization. http://apps.who.int/iris/bitstream/10665/85975/1/WHO_NMH_MND_13.2_eng.pdf.

WHO criteria for use of HbA1c.

Diabetes websites

American Diabetes Association. http://www.diabetes.org.

Diabetes UK. http://www.diabetes.org.uk.

World Health Organization. Diabetes. http://www.who.int/topics/diabetes_mellitus/en/.

Which type of diabetes is it?

Table 2.17 shows the types and causes of diabetes. Type 2 diabetes due to insulin resistance is by far the commonest (accounting for >90% of cases) and is ↑ as the prevalence of obesity and low physical activity in our society rise. However, there are no definitive tests that can distinguish type 1 and type 2 diabetes. Instead, a collection of clinical and laboratory parameters are used (see Table 2.18), but many cases are hard to categorize (sometimes referred to as type 1.5 diabetes!).

Table 2.17 Types and causes of diabetes

Type of diabetes*	Comment
Type 1	β-cell destruction usually leading to absolute insulin deficiency: 1A—immune-mediated; 1B—idiopathic (formerly known as juvenile-onset or IDDM)
Type 2	Predominantly insulin-resistant with relative insulin deficiency (formerly known as maturity-onset or NIDDM)
Gestational diabetes	Diabetes during pregnancy that resolves postpartum. Often an early manifestation of type 2 diabetes
Other specific types	Includes genetic (MODY, mitochondrial, insulin resistance syndromes), pancreatic disease, drug-induced, occurrence in other genetic syndromes, endocrinopathies (e.g. acromegaly)

* Abbreviated from the ADA classification.

Figure 2.18 (guide to the type of diabetes) provides an algorithm for diagnosing the type of diabetes. Although type 2 diabetes remains the commonest diagnosis in adults and is increasingly common in children, this is a diagnosis for the lifetime of the individuals and care should be taken to diagnose any underlying conditions as accurately as possible. Note especially:

•Unusual features: if any of the unusual features listed in Table 2.19 are present, then the algorithm should not be pursued and an underlying cause for the diabetes investigated.

•Type 1 diabetes: it is important to always consider the possibility of type 1 diabetes (see Fig. 2.18), even in older people, as early insulin treatment is essential to avoid the risk of ketoacidosis.

•Pregnancy: although the majority of diabetes diagnosed for the first time in pregnancy (gestational diabetes) is part of the spectrum of type 2 diabetes, diabetes due to other causes can occur. The algorithm in Fig. 2.18 should still be considered.

•Latent autoimmune diabetes of adults (LADA) refers to diabetes with +ve autoantibodies (anti-glutamic acid decarboxylase (GAD)) developing in adults (typically over the age of 35) but not developing ketosis or with absolute requirement for insulin within 6 months of diagnosis. However, subjects are usually not overweight and develop a need for insulin within a few years. LADA is considered to be a ‘slow-onset’ version of type 1 diabetes, often initially misdiagnosed as type 2 diabetes.

Table 2.18 Clinical features and laboratory tests used to distinguish type 1 and type 2 diabetes, and maturity-onset diabetes of the young (MODY)

		Type 1	Type 2	MODY
Clinical features	Weight	Slim (BMI <25) + weight loss at diagnosis	Overweight (BMI >25)	Average weight (BMI <30)
	Ketosis	Occurs	Rare	Rare
	Race	Caucasian	↑ risk in South Asians and Afro-Caribbeans
	Acanthosis nigricans	Absent	May be present	Absent
	Parent with diabetes	Unusual	Common	Common
Laboratory tests	Auto-antibodies	Anti-GAD antibody +ve in around 80%*	−ve	−ve
	Insulin c-peptide	Low (but still present for up to 5 years from diagnosis)	Present	Present
	High-density lipoprotein (HDL) >1.2	Usual	Rare	Usual

* Additional antibody tests can include anti-IA2 and anti-ZnT8 antibodies.

•Monogenic diabetes—maturity-onset diabetes of the young (MODY): monogenic disorders resulting in diabetes (see Tables 2.19 and 2.20). Although these account for <1% of cases, they are important to diagnose as they may be easily treated with sulfonylureas (MODY 3), associated with renal disease (MODY 5), or require no treatment (MODY 2). The diagnosis also has important implications for other family members diagnosed with diabetes. Note that it can be very difficult to distinguish type 2 diabetes from MODY without genetic testing, and a high level of suspicion in young people is required (see Fig. 2.18). If suspected, further advice from a genetic testing centre with experience in MODY should be sought ( http://www.diabetesgenes.org).

•‘Flatbush’ diabetes refers to patients who present in DKA but subsequently have a course that is more like type 2 diabetes and are able to come off insulin. This is most commonly seen in African-Caribbeans.

Fig. 2.18 Guide to the type of diabetes.

Table 2.19 Specific features suggestive of an unusual cause of diabetes

Feature	Possible diagnosis
Alcohol excess, history of pancreatic disease	Chronic pancreatitis
Painless jaundice, weight loss in older person	Pancreatic cancer
Cystic fibrosis	Pancreatic disease
On steroids/Cushingoid appearance	Steroid-induced diabetes/Cushing’s syndrome
Post-organ transplant	Drug-induced
Specific drugs, e.g. somatostatin analogues, diazoxide	Drug-induced
Severe hypertension	Phaeochromocytoma
Acromegalic appearance	Acromegaly
‘Muscular appearance’ (lipodystrophy)	Partial lipodystrophy
Family history of diagnosis <25 in two or more generations	MODY
Renal cysts, urogenital dysplasia	Hepatic nuclear factor (HNF)1β MODY
Bilateral deafness (maternal inheritance)	Mitochondrial diabetes
Optic atrophy, DI	Wolfram syndrome
Diagnosis <6 months old	Kir 6.2 mutation or other forms of neonatal diabetes
Megaloblastic anaemic	Roger’s syndrome (thiamine)
Short stature, severe insulin deficiency (>1000U/day)	Insulin receptor defect (leprechaunism, Rabson–Mendenhall syndrome)
Stiff legs/gait/falls: −ve-GAD antibodies	Stiff person syndrome

Table 2.20 Genetically inherited forms of diabetes, including MODY

Gene	% of MODY cases	Comments
Glucokinase	20	MODY 2 ‘mild’ complications rare
HNF-1α	60	MODY 3 diagnosed later—35 years, progressive β cell failure, but very sensitive to sulfonylureas
HNF-4α	1	MODY 1
HNF-1β	1	MODY 5 renal cysts/abnormalities
IPF-1	1	MODY 4
NeuroD1	? <1	MODY 6
Unknown	15	‘MODY X’
SUR1	<1%??	Hyperinsulinism in infancy and β cell failure as adult
Mitochondrial	Not MODY	Maternally inherited. May be associated with nerve deafness, lactic acidosis, or syndromes such as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness)
Lipodystrophy	Not MODY, lamin a/c and others	Associated with localized fat loss

Further reading

DiabetesGenes.org. http://www.diabetesgenes.org.

Monitoring diabetic control

Self-testing and near-patient testing

Self-testing capillary blood glucose (or urine) can be readily performed by the majority of patients, with results available in under 20s. Measurements of longer-term glycaemic control are typically laboratory-based, although increasingly near-patient testing equipment is available for use in clinics that can give results to patients within minutes.

Urine testing

Glycosuria

Semi-quantitative testing for glucose using reagent-impregnated test strips is of limited value and although used to ‘screen’ for diabetes, it is not recommended for monitoring of glycaemic control. Urinalysis provides retrospective information over a limited period of time. Other limitations are:

•The renal threshold for the reabsorption of glucose in the proximal convoluted tubule (PxCT) is ~10mmol/L on average but varies between individuals. Subjects with a low threshold will tend to show glycosuria more readily, even with normal glucose tolerance (‘renal glycosuria’). Children are particularly liable to test +ve for glucose. The renal threshold is effectively lowered in pregnancy. Conversely, a high threshold, common among the elderly, may give a misleadingly reassuring impression of satisfactory control. Fluid intake and urine concentration may affect glycosuria. Renal impairment may elevate the threshold for glucose reabsorption.

•Delayed bladder emptying, e.g. due to diabetic autonomic neuropathy, will reduce the accuracy of the measurements through dilution.

•Hypoglycaemia cannot be detected by urinalysis.

Ketonuria, self-blood testing for ketones

Semi-quantitative test strips for acetoacetate (e.g. Ketostix^®) are available for patients with type 1 DM but have largely been replaced by self-testing of blood (capillary, fingerprick) for ketones (β-hydroxybutyrate). Useful when intercurrent illness leads to disturbance of metabolic control. The presence of ketonuria on dipstick testing (++ or +++) or blood ketones (>3.0mmol/L) in association with hyperglycaemia indicates marked insulin deficiency. The patient may be developing ketoacidosis, and ↑ insulin doses and urgent assessment for DKA is required ( Diabetic emergencies: diabetic ketoacidosis, hyperosmolar non-ketotic syndrome, and lactic acidosis, pp. 210–211). Note that low-level ketonuria (‘+’) or blood ketones <0.6mmol/L can occur after a period of fasting, especially in overweight patients, and does not necessarily indicate DKA. Occasionally, patients with type 2 DM develop ketosis during severe intercurrent illness, e.g. major sepsis. Urine testing strips do not detect 3-hydroxybutyrate (although acetone is detected by Acetest^®). Occasional underestimation of the degree of ketonaemia using these tests is a well-recognized, albeit uncommon, caveat of alcoholic ketoacidosis but is no longer an issue with the use of blood ketone testing. Blood testing is preferred, as it does not require any delay in obtaining a urine sample and is more accurate. Separate testing strips from glucose testing are required (but often the same meter can be used).

Self-testing of blood glucose

Self-testing of capillary blood glucose obtained by fingerprick has become an established method for monitoring glycaemic control. Frequent testing is a prerequisite for adjusting insulin doses and for safe intensive insulin therapy such as that employed in the DCCT. Use in type 2 diabetes treated by diet or tablets only is not essential. Enzyme-impregnated dry strip methods are available, which are used in conjunction with meter devices and give results in <20s with just 50µL of blood. Adequate training and a system of quality control are important; even when trained health professionals use such systems in clinics or hospitals, misleading results are possible, particularly in the lower range of blood glucose results. Where there is doubt, an appropriate sample (in a tube containing the glycolysis inhibitor fluoride oxalate) should be collected immediately for analysis by the clinical chemistry laboratory. However, acute treatment of hypoglycaemia, where indicated, should not be delayed.

Continuous glucose monitoring systems (CGMS)

Several systems are now available using electrical conductance or microdialysis to provide continuous monitoring of interstitial glucose. Sensors are inserted SC and need to be replaced every 3–7 days. Most systems can display the results in real time (if regularly calibrated against traditional finger stick readings), so that they can be reviewed by the patient, and linked to alarms indicating high and low levels. While these systems, although expensive, are proving increasingly valuable for patients with type 1 diabetes on complex insulin regimes and insulin pump therapy, it must be remembered that the interstitial glucose level is up to 30min ‘behind’ the blood level and if glucose levels are changing rapidly, continuous monitors may ‘miss’ significant hypoglycaemic events. A recent development is sensors that do not require calibration and the reading is made by ‘swiping’ the reader (or an appropriately configured smartphone) over the sensor. These newer sensors have significantly reduced ‘delay’ time, claimed to be <10min vs blood levels.

Laboratory assessment of glycaemic control

Glycated haemoglobin

Measuring HbA_1c

HbA_1c (comprises 60–80% of total glycated haemoglobin HbA₁) is formed by the slow, irreversible post-translational non-enzymatic glycation of the N-terminal valine residue of the β chain of Hb. The proportion of HbA_1c:total Hb (normal non-diabetic reference range ~4–6%) provides a useful index of average glycaemia over the preceding 6–8 weeks. The result is disproportionately affected by blood glucose levels during the final month before the test (~50% of value). Laboratory values have now been aligned to a standard from the DCCT trial (DCCT-aligned), and values are generally consistent between laboratories. Recent recommendations suggest expressing HbA_1c in new units (mmol/mol) against a new International Federation of Clinical Chemistry (IFCC) standard (see Table 2.21).

Table 2.21 Guide to HbA_1c values in new and old units

DCCT-aligned HbA_1c (%)	IFCC-HbA_1c (nmol/mol)
6.0	42
6.5	48
7.0	53
7.5	59
8.0	64
9.0	75

Frequency of testing HbA_1c

It is suggested that HbA_1c is measured every 6 months in stable patients, every 3 months in patients with unstable metabolic control, and every month in pregnancy.

Interpreting HbA_1c levels

Average HbA_1c levels collected over a longer period (i.e. years) provide an estimate of the risk of microvascular complications. Sustained high concentrations identify patients in whom efforts should be made to improve long-term glycaemic control and ↑ surveillance for long-term complications. Table 2.22 summarizes recent recommendations for target HbA_1c levels and capillary glucose measurements in adult subjects with diabetes. Targets need to be modified in pregnancy (see footnote to Table 2.22), in children, in patients with recurrent hypoglycaemia or difficulty complying with medication, and in patients with vascular (especially coronary artery) disease in whom hypoglycaemia may precipitate fatal arrhythmias.

Table 2.22 Target levels of HbA_1c and capillary blood glucose values

	Pre-meal (mmol/L)	Post-meal (mmol/L)	HbA_1c (nmol/mol)
Non-diabetic	3.5–5.5	<7.8	4–6% (20–42)
Ideal	4–7*	<10*	<7%** (<53)
Suboptimal control			7–10%
Poor control			>10%

* In pregnancy, NICE recommends 3.5–5.9 pre-meal and <7.8 post-meal (1h) HbA_1c; aim for <6.1%.

** In type 2 diabetes, NICE recommends a target of 6.5% in patients without vascular disease.

Note: lower target levels (HbA_1c <6.5%) have been proposed in subjects with type 2 diabetes treated with lifestyle or metformin alone; Higher targets (e.g. HbA_1c <8.0% or higher) are appropriate in subjects with frequent hypoglycaemia or hypoglycaemia unawareness, extensive co-morbid conditions, or limited life expectancy.

Limitations of HbA_1c measurements

Although glycated Hb levels are a reliable indicator of recent average glycaemic control, they do not provide information about the daily pattern of blood glucose levels or the frequency of hypoglycaemic episodes; this supplementary information required for logical adjustment of insulin doses is derived from frequent home blood glucose monitoring. More recent changes in glycaemia (i.e. within the preceding 4 weeks or so) will influence HbA_1c level more than glucose levels 12 or more weeks ago.

Spurious HbA_1c levels may arise in states of:

•Blood loss/haemolysis/reduced red cell survival (low HbA_1c).

•Haemoglobinopathy.

•↑ levels of HbS (low levels of HbA_1c)

•↑ levels of HbF (high HbA_1c).

Modern HbA_1c methods are likely to detect haemoglobinopathies without specific testing. Where haemoglobinopathy is present and cannot be adjusted for by an assay method where there is no interference, the HbA_1c test is uninterpretable and capillary blood glucose levels or fructosamine must be used.

HbA_1c measurements are less reliable in pregnancy where rapid changes in blood glucose levels can occur (e.g. last trimester). They are still used, as they are more reliable than other available methods or estimating overall control, but results should be interpreted with caution.

Uraemia due to advanced diabetic nephropathy is associated with anaemia and ↓ RBC survival, thereby falsely lowering HbA_1c levels.

Fructosamine: refers to protein–ketoamine products resulting from the glycation of plasma proteins. The fructosamine assay measures glycated plasma proteins (mainly albumin), reflecting average glycaemia over the preceding 2–3 weeks. This is a shorter period than that assessed using glycated Hb measurements and may be particularly useful when rapid changes in control need to be assessed, e.g. during pregnancy. Levels can be misleading in hypoalbuminaemic states, e.g. nephrotic syndrome. Some fructosamine assays are subject to interference by hyperuricaemia or hyperlipidaemia.

The main indications for fructosamine measurement are currently: (a) the presence of haemoglobinopathy or other interference with the HbA_1c assay ( Limitations of HbA_1c measurements, pp. 207–208) and (b) rapidly changing blood glucose levels (e.g. pregnancy).

Further reading

American Diabetes Association (ADA). Clinical guidelines on pages for health professionals. http://www.diabetes.org.

American Diabetes Association. 5. Glycemic targets. Diabetes Care 2016; 39(Suppl 1), S39–46 (ADA standards of medical care).

National Institute for Health and Care Excellence. Diabetes guidelines. http://www.nice.org.uk.

Diabetic emergencies: diabetic ketoacidosis, hyperosmolar non-ketotic syndrome, and lactic acidosis

DKA should be considered in any unconscious or hyperventilating patient. The hyperosmolar non-ketotic (HONK) syndrome is characterized by marked hyperglycaemia (>30mmol/L) and dehydration in the absence of significant ketosis or acidosis. Lactic acidosis (LA) associated with metformin is uncommon. A rapid clinical examination and bedside blood tests should allow the diagnosis to be made. Treatment (IV rehydration, insulin, electrolyte replacement) of these metabolic emergencies should be commenced without delay (for details, Further reading, p. 211).

Confirm diagnosis by bedside measurement of

•Capillary blood glucose.

•Capillary blood testing for ketones (betahydroxybutyrate).

•Urine for nitrites and leucocytes (UTI).

Venous blood for urgent laboratory measurement of

•Plasma glucose (fluoride oxalate; ► true ‘euglycaemic’ DKA is rare).

•U&E (arterial potassium (K⁺) can be measured by some gas analysers). Plasma Na⁺ may be depressed as a consequence of hyperglycaemia or marked hyperlipidaemia.

•Plasma creatinine (► may be falsely elevated in some assays by DKA).

•Plasma lactate (if indicated—can also be measured by some gas analysers). Indicated if acidosis without heavy ketonuria is present. LA is a complication of tissue hypoxia (type A) and is a rare complication of metformin treatment in patients with type 2 DM (type B).

•Plasma osmolality in HONK—either by freezing point depression or calculated:

• 2 × [plasma Na⁺] + [plasma K⁺] + [plasma glucose] + [plasma urea]

•FBC (non-specific leucocytosis is common in DKA).

•Blood cultures (signs of infection, e.g. fever, may be absent in DKA).

•ABGs (corrected for hypothermia) for arterial pH, HCO₃⁻, PCO₂, and PO₂ (if shock or hypotension).

DKA is confirmed by blood glucose >11mmol/L (rarely euglycaemic ketoacidosis can occur in pregnancy or subjects on sodium–glucose cotransporter 2 (SGLT2)-inhibiting drugs), ketonaemia >3.0mmol/L, and acidosis (pH <7.3 or HCO₃⁻ <15mmol/L). Severe DKA is considered to be pH <7.0, HCO₃⁻ <5mmol/L, or ketones >6.0mmol/L. Repeat laboratory measurement of blood glucose, electrolytes, and urea at 2, 4, and 6h, and as indicated thereafter. Electrolyte disturbances, renal impairment, or oliguria should prompt more frequent (1–2h) measurements of plasma K⁺. Capillary blood glucose and ketone testing are monitored hourly at the bedside. ► Avoidance of hypokalaemia and hypoglycaemia is most important during therapy. Current therapy is aimed at rapid resolution of ketosis which is considered to be ketonaemia <0.6mmol/L (‘normal’ <0.3mmol/L) with pH >7.3.

Other investigations, as indicated

•CXR.

•Microbial culture of urine, sputum, etc.

•ECG: acute MI may precipitate metabolic decompensation; note that serum transaminases and CK may be non-specifically elevated in DKA.

•Sickle-cell test (in selected patients).

•Venous plasma PO₃⁴⁻ (if there is respiratory depression).

•► Performance of investigations should not delay initiation of treatment and transfer to a high-dependency or intensive care unit (ICU).

Severe metabolic acidosis in the absence of hyperglycaemia (or other obvious cause of acidosis such as renal failure) raises the possibility of

•LA.

•Alcoholic ketoacidosis: this occurs in alcoholics following a binge. Alterations in the hepatic redox state may result in a misleading −ve or ‘trace’ Ketostix^® reaction but detectable on blood ketone strips. A similar caveat may occasionally be encountered when significant LA coexists with DKA. Venous plasma glucose may be normal or ↑.

► Anion gap >15mmol/L. Normally, the anion gap (<10mmol/L) results from plasma proteins, sulfate (SO₄²⁻), PO₄³⁻, and lactate ions. When the anion gap is ↑, measurement of plasma ketones, lactate, etc. usually confirms the aetiology (see Table 2.23).

Table 2.23 Causes of an anion gap acidosis

Ketoacidosis	DKA
	Alcoholic ketoacidosis
	LA (► metformin)
Chronic renal failure
Drug toxicity	Methanol (metabolized → formic acid)
	Ethylene glycol (metabolized → oxalic acid)
	Salicylate poisoning

Further reading

Joint British Diabetes Societies (JBDS) guidelines (2013). Available at: http://www.diabetologists-abcd.org.uk.

Investigation of hyperlipidaemia

1° dyslipidaemias are relatively common and contribute to an individual’s risk of developing atheroma (e.g. CHD, CVD). Prominent examples include familial combined hyperlipidaemia (FCHL; ~2–3% of UK population) and heterozygous familial hypercholesterolaemia (FH; UK incidence 1 in 500). Major hypertriglyceridaemia also predisposes to pancreatitis. The key features of familial FH, FCHL, and diabetic dyslipidaemia are considered later.

Investigations

Although many subtle alterations in plasma lipids have been described, therapeutic decisions rest on measurement of some or all of the following in serum or plasma (plasma being preferred, since it can be cooled rapidly):

•Total cholesterol (may be measured in non-fasting state in first instance, since levels are not greatly influenced by meals).

•Triglycerides (TGs) (after 12h fast).

•LDL-cholesterol (calculated using the Friedewald formula when TGs are <4.5mmol/L):

Note that because of the unreliability of this calculation, especially at higher TG levels, it has been recommended to report results also as ‘non-HDL-cholesterol’, which is typically around 0.7mmol/L higher than calculated LDL-cholesterol.

•HDL-cholesterol (regarded as the ‘cardioprotective’ subfraction)—HDL particles are synthesized in the gut and liver and thought to be involved in ‘reverse transport’ of cholesterol from peripheral tissues to the liver where it can be excreted as bile salts.

Notes on sampling in relation to lipoprotein metabolism

•TGs (triacylglycerols) are measured after a 712h overnight fast in order to clear diet-derived chylomicrons.

•Alcohol should be avoided the evening prior to measurement of TGs (can exacerbate hypertriglyceridaemia).

•A weight-maintaining diet is recommended for 2–3 weeks before testing.

•Lipid measurements should be deferred for 2–3 weeks after minor illness and 2–3 months after major illness, surgery, or trauma since cholesterol levels may be reduced. Following acute MI, it is generally accepted that plasma cholesterol is reliable if measured within 24h of the onset of symptoms.

•The effects of certain drugs on lipids should be considered (see Box 2.10).

•Glycaemic control should be optimized wherever possible before measuring plasma lipids in patients with diabetes.

Important additional considerations are

•Day-to-day variability: generally, decisions to treat hyperlipidaemia should be based on >1 measurement over a period of 1–2 weeks. This is especially true for patients with mixed hyperlipidaemia (i.e. including hypertriglyceridaemia).

•Exclusion of 2° hyperlipidaemia—many common conditions, drugs, and dietary factors can influence plasma lipids (see Box 2.10).

•Family members should also have their plasma lipids measured if familial hyperlipidaemia is suspected in a proband.

Both cholesterol and TGs may be affected to some degree by these factors, but one often predominates. Pre-existing 1° hyperlipidaemias may be exacerbated.

Clinical features

For example, xanthelasma, tendon xanthomas, etc. should always be sought. A detailed family history, drug history, and medical history (for diabetes and other cardiovascular risk factors such as hypertension) should always be obtained. Certain endocrine disorders and impaired hepatic or renal function can influence circulating lipid composition and cardiovascular risk. A classification of the major familial dyslipidaemias is presented in Table 2.24.

Box 2.10 Causes of secondary hyperlipidaemia

Hypercholesterolaemia

•Hypothyroidism (even minor degrees of 1° hypothyroidism)

•Cholestasis (raised lipoprotein X levels)

•Nephrotic syndrome

•Anorexia nervosa

•Diuretics

•Immunosuppressive agents

•Hepatoma

•Dysglobulinaemias

Hypertriglyceridaemia

•Obesity

•Diabetes (especially type 2 DM)

•Lipodystrophic syndromes (of diabetes and HIV-associated)

•Alcohol excess (note: moderate alcohol consumption may raise HDL-cholesterol)

•Renal failure

•Antiretroviral agents

•Oestrogens (especially oral preparations in some women)

•Corticosteroids

•β-adrenergic blockers

•Retinoids

Recommended investigation for exclusion of 2° hyperlipidaemia: U&E, plasma creatinine, fasting venous glucose, LFTs, and TFTs. For patients on statins, check LFTs and CK periodically (► measure urgently if myositis occurs—a rare, but potentially fatal, complication).

Table 2.24 Familial hyperlipoproteinaemias

Genetic disorder	Defect	Presentation	Cholesterol	Triglycerides	Phenotype
Familial LPL deficiency	Absence of LPL activity	Eruptive xanthomata; hepatosplenomegaly	↑	↑↑↑	I
Familial apo C-II deficiency	Absence of apo C-II	Pancreatitis	↑	↑↑↑	I or V
Familial hypercholesterolaemia	LDL receptor deficiency	Tendon xanthomata; premature atheroma	↑↑↑	↑ or N	IIa or IIb
Familial dysbeta-lipoproteinaemia	Abnormal apo E and defect in TG metabolism	Tubo-eruptive and palmar xanthoma; premature atheroma	↑↑↑	↑↑↑	III
Familial combined hyperlipidaemia	Uncertain	Premature atheroma	↑ or N	↑ or N	IIa, IIb, or IV
Familial hypertriglyceridaemia	Uncertain; eruptive xanthomata; hepatosplenomegaly; pancreatitis		N		IV
Familial hypertriglyceridaemia			↑	↑↑↑	V

↑, ↑↑, and ↑↑↑, mildly, moderately, and serverly raised; respectively cholesterol and triglycerides refer to concentrations in plasma; phenotype refers to Fredrickson classification (I to V, see Table 2.25); apo, apoprotein; LPL, lipoprotein lipase; n, normal; TG, triglycerides.

► Specialist advice should be sought in the management of major or resistant hyperlipidaemias. TG levels >11mmol/L (1000mg/dL) are considered ‘very high’ and require therapy because of the risk of pancreatitis.

Table 2.25 Phenotypic (Fredrickson) classification of hyperlipidaemias

Type	Cholesterol	Triglycerides	Particle excess	Usual underlying cause
I	↑	↑↑↑	Chylomicrons	LPL or apo C-II deficiency
IIa	↑↑	↑	LDL	LDL receptor defect, LDL overproduction
IIb	↑↑	↑↑	VLDL, LDL	VLDL or LDL overproduction or ↓ clearance
III	↑↑	↑↑	IDL— dysbetalipoproteinaemia	Impaired remnant removal may be due to certain apo E phenotypes or apo E deficiency
IV	N or ↑	↑↑	VLDL	VLDL overproduction or ↓ clearance
V	↑	↑↑↑	ChylomicronsVLDL	Diabetes

↑, ↑↑, and ↑↑↑, mildly, moderately, and severely raised, respectively; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein; LPL, lipoprotein lipase; apo, apoprotein; IDL, intermediate-density lipoproteins.

Further reading

JBS3 Board. Joint British Societies’ consensus recommendations for the prevention of cardiovascular disease (JBS3). Heart 2014; 100 Suppl 2: ii1–67.

National Heart, Lung, and Blood Institute, National Cholesterol Education Programme Guidelines (2013). Managing blood cholesterol in adults: systematic evidence review from the Cholesterol Expert Panel. http://www.nhlbi.nih.gov/health-pro/guidelines/in-develop/cholesterol-in-adults.

Test protocols

Insulin tolerance test (insulin stress test)

•Indication: suspected ACTH or GH deficiency.

•Contraindications: patients with epilepsy, CHD (check ECG).

•Children: use no more than 0.1U/kg. Considerable care should be exercised; the test should only be performed in a centre with expertise.

•Alternatives: short Synacthen^® test for hypocortisolism; stimulation tests for GH deficiency, e.g. growth hormone-releasing hormone (GHRH) arginine test ( Hypothalamus/pituitary function, see pp. 128–130).

•Preparation: patient fasting overnight. Bed required (although day-case procedure). Patient must be accompanied home and may not drive. OMIT morning hydrocortisone or other steroid hormone replacement if patient is taking this and previous day’s GH. Physician must be present throughout the test. Requires written consent.

•Procedure: early morning outpatient test in fasting patient. Indwelling venous cannula and constant medical supervision required throughout. Cannula is kept patent by running a saline infusion with a three-way tap for sampling. Discard initial 2–3mL when each sample is taken. Label all samples clearly with time and patient details. Near-patient testing glucometer required.

1.Take baseline blood for glucose, cortisol, and GH. Check IV access is working well. Review the test with the patient, and explain symptoms s/he is likely to experience (see point 5).

2.Draw up 25mL of 50% glucose for immediate administration IF REQUIRED.

3.Give soluble (regular) insulin as an IV bolus in a dose of 0.15U/kg after an overnight fast. Consider 0.1U/kg (lower dose) if suspected profound hypocortisolism. ► This appears a very small dose, e.g. typically around 10U. CHECK DOSE CALCULATION CAREFULLY. Usually an insulin syringe is used to draw it up and then transfer it to a 2mL syringe containing saline.

4.Take blood at 15min intervals (0, 15, 30, 45, 60min) for glucose, cortisol, and GH.

5.Observe for symptoms and signs of hypoglycaemia. The first sign is usually profuse sweating. The patient may then be aware of symptoms such as palpitations, hunger, and paraesthesiae. This typically occurs 30–45min into the test. Check near-patient glucose to confirm <3.5mmol/L. Continue to talk to, and reassure, the patient. If the patient becomes very drowsy or unrousable, then give 25mL of 50% glucose. This does not invalidate the test, as the hypoglycaemic stimulus has already occurred. Continue blood sampling at standard times.

6.If the patient has not experienced hypoglycaemia by 45min and near-patient glucose is >4mmol/L, give a further IV bolus of 0.15 or 0.3U/kg if the patient is known to be very insulin-resistant (e.g. acromegalic). Repeat sampling at 15min intervals for 60min after this second bolus.

7.At the end of procedure (usually 60min), give IV 25mL of 50% dextrose if the patient still has symptoms of hypoglycaemia.

8.Give the patient a meal including complex carbohydrate (e.g. sandwiches or lunch), and observe for a minimum of 1h further before accompanied discharge.

•Unwanted effects: severe hypoglycaemia with depressed level of consciousness or convulsion requires immediate termination of the test with 25mL of 50% glucose IV. Repeat if necessary and follow with 5% or 10% glucose infusion. Continue to collect samples for hormone and glucose measurements.

•Interpretation: the test is only interpretable if adequate hypoglycaemia is achieved (<2.2mmol/L). Normal maximal cortisol response >550nmol/L. (Note: may be lower, depending on local assay range—check with the laboratory). Normal GH response >20mU/L. Impaired responses (if hypoglycaemic stimulus adequate) denote corticotrophin (assuming adrenal glands are normal) or GH deficiency or both. Peak GH response <10mU/L is sufficient to consider GH replacement; peak GH response <5mU/L is severe GH deficiency.

Combined arginine–growth hormone-releasing hormone test

•Indication: GH deficiency. Now preferred to ITT.

•Contraindications: previous reaction to stimulatory hormones. Administer with caution to patients with severe liver or renal disease.

•Alternatives: ITT. Other stimulation tests now outdated (e.g. glucagon, exercise). Serum IGF-1 levels give an idea of the GH status but are unreliable at low levels.

•Preparation: order GHRH and arginine from pharmacy. Omit GH injections for a minimum of 24h. The patient arrives in the morning after fasting for 10h (overnight). Water is allowed and patients should take all their routine medications in the morning (but not GH). Informed consent must be obtained and documented. Warn patients about possible side effects of IV GHRH such as flushing lasting <5min.

•Procedure:

•Weigh the patient.

•Insert an indwelling IV cannula for blood sampling, administration of bolus GHRH and arginine infusion (keep patent with heparinized saline).

•Patients should rest throughout the test.

•Take basal samples for GH at −30 and 0min.

•Give GHRH as an IV bolus of 1µg/kg body weight at 0min; at the same time, start infusing 30g of 12.5% arginine solution over 30min, preferably using an infusion pump (children: 0.5g/kg as a 12.5% solution, to a maximum of 30g).

•Take blood samples for GH at 30, 45, 60, 75, 90, 105, and 120min.

•Patients are allowed home after a full lunch.

•Interpretation: the diagnosis of adult GH deficiency is confirmed if the peak GH concentration is <15mIU/L (by ITT, <12mU/L in GHRH testing). Severe GH deficiency (as defined by NICE)—peak <9mIU/L. Cut-offs by BMI range have been proposed (34.5mU/L for those with a BMI <25kg/m², 24mU/L for a BMI of 25–30kg/m², and 12.6mU/L for those with a BMI >30kg/m²) but are not universally accepted.

Note: conversion factor: µg/L × 3.0 = mIU/L.

Combined anterior pituitary function testing

•Indication: assessment for anterior pituitary hypofunction.

•Contraindications: previous reaction to stimulatory hormones.

•Alternatives: ITT for GH and adrenal axis; metyrapone test for adrenal axis. Other stimulation tests for GH, e.g. GHRH–arginine test.

•Preparation: test usually performed in morning for basal sampling.

•Procedure: IV cannula inserted. Basal blood samples taken for cortisol, oestradiol (♀) or testosterone (♂), FT4, and IGF-1. Hypothalamic hormones are given sequentially IV, each as a bolus, over around 20s: LHRH 100µg, TRH 200µg, and ACTH 250µg. Additionally, GHRH (1µg/kg body weight) may be given. (Reduce doses in children.) Samples are drawn at 0, 20, 30, 60, and 120min for LH, FSH, TSH, cortisol, and PRL. If GHRH is given, samples are drawn at the same time points for GH.

•Interpretation: normal values as follows:

•TRH—suspect 2° hypothyroidism if peak response (at 20min) <20mU/L. (Note: low levels also seen in hyperthyroidism—ensure FT4 or total T4 not raised.)

•ACTH—peak cortisol response >550nmol/L at 30 or 60min. (Note: may be lower, depending on local assay range—check with the laboratory.)

•LHRH—peak LH/FSH response 2–5 times the basal value. LH, peak at 20min; FSH later.

•GHRH—normal GH peak response >15mU/L.

Water deprivation test

•Indication: diagnosis of DI and to distinguish cranial and nephrogenic DI.

•Contraindications: none if carefully supervised. For correct interpretation, thyroid and adrenal deficiencies should be replaced first. Interpretation in the presence of DM and uraemia can be difficult.

•Alternatives: morning urine osmolality of >600mOsmol excludes significant degrees of DI. No other definitive test for DI.

•Patient preparation: usually an outpatient procedure. Correct thyroid and adrenal insufficiencies in advance. Renal function and blood glucose should have been checked in advance. Steroid and thyroid hormone replacement should be taken as normal on the day of the test. If the patient is on desmopressin, omit the dose on the evening before the test (or, if not possible, halve this dose). Free fluids, but not to excess, up to 7.30 a.m. on the day of the test. No alcohol on the night before the test or on the morning of the test. Light breakfast, but no tea, coffee, or smoking on the morning of the test. Empty the bladder before attending for the test. If urine volume is <3L/day (‘mild cases’), ask the patient to have no fluids or food from 6.00 p.m. on the evening before the test (‘prolonged water deprivation test’).

•Requirements for test: accurate weighing scales. Supervision for the whole test (up to 8h). Desmopressin for injection (2µg). Immediate access to serum electrolyte, plasma, and urinary osmolality assays. Access to a plasma arginine vasopressin (AVP) (ADH) assay desirable.

•Procedure: 7.30 a.m.

1.Weigh the patient, and calculate 97% of the body weight.

2.Mark this target on the chart.

3.No food or fluid for the next 8h.

4.Insert a cannula for repeated blood sampling, and flush.

•Procedure: 8 a.m.

5.Obtain plasma for Na⁺ and osmolality, and urine for osmolality.

6.Then collect urine hourly for volume and osmolality, and plasma every 2h for Na⁺ and osmolarity.

7.Weigh the patient before and after passing urine if unobserved.

8.If patient loses 3% of the body weight, order urgent plasma osmolality and Na⁺.

9.If plasma osmolality >300mOsmol (Na⁺ >140mmol/L), stop the test; allow the patient to drink, and give desmopressin (see point 14).

10.If plasma osmolality <300mOsmol, the patient may have been fluid-overloaded before the test, and water deprivation can continue.

11.Stop the test at 8h (4.00 p.m.), and take final recordings of urine and plasma.

12.Save an aliquot of plasma for vasopressin levels in case of difficulties in test interpretation.

13.Ideally urine osmolalities will have reached a plateau (<30mOsmol rise between samples).

14.Now give 2µg of desmopressin intramuscularly (IM) (or 20µg intranasally), and collect urine samples only for a further 2h. Allow free fluids at this stage.

•Interpretation: normal response: plasma osmolality remains in the range of 280–295mmol; urine osmolality rises to >2 times plasma (>600mOsmol). If urine volumes during water deprivation do not reduce and yet the plasma does not become more concentrated (rising osmolality) and weight does not fall, suspect surreptitious drinking during the test. For interpretation of abnormal results, Table 2.2, p. 136

Diagnostic trial of desamino d -arginyl vasopressin

•Indication: distinction of partial DI from 1° polydipsia.

•Contraindications: cardiac failure; current diuretic use (test uninterpretable). Note that this test may precipitate severe hyponatraemia in 1° polydipsia and should be performed in an inpatient unit with clinical and biochemical regular review.

•Preparation: admission to an assessment unit. First-line tests for polydipsia/polyuria should have been performed ( Polydipsia and polyuria: diabetes insipidus, pp. 134–136).

•Procedure:

1.24h urine volume, morning urine osmolality, weight, fluid intake (as far as possible), serum osmolality, Na⁺, urea, and creatinine should all be performed daily and the results reviewed the same day.

2.Subjects should have access to fluid ad libitum but should be reminded that they should only drink if they are thirsty.

3.After an initial 24h period of observation, desmopressin is administered at a dose of 2µg bd SC for 3 days.

4.Stop the test if serum Na⁺ falls to <130mmol/L.

•Interpretation: reduction in urine volume to <2L/day and in urine osmolality to >600mOsmol/L without a fall in serum Na⁺ to <140mmol/L suggests central DI. Reduction in urine volume with no ↑ in urine osmolality >600mOsmol/L and without a fall in serum Na⁺ suggests partial nephrogenic DI. Limited reduction in urine volume, with some ↑ in urine osmolarity, but a fall in serum Na⁺, suggests 1° polydipsia.

Low-dose dexamethasone suppression test

•Indication: to distinguish hypercortisolism from normality. The dexamethasone-suppressed CRH test is believed to have less false +ves in cases of alcoholic or depressive pseudo-Cushing’s syndrome.

•Patient preparation: patients should not be on oral steroids or drugs that ↑ steroid metabolism.

•Overnight dexamethasone suppression test: 1mg of dexamethasone is taken by mouth (PO) at midnight. A serum sample for cortisol is taken the following morning between 8 a.m. and 9 a.m.

•Interpretation: serum cortisol should suppress to <140nmol/L (usually <50nmol/L). Values of 140–175nmol/L are equivocal and suggest a 2-day test should be performed. There is 10–15% false +ve rate.

•Two-day low-dose dexamethasone suppression test (preferred): dexamethasone 0.5mg is given PO every 6h for eight doses (2 days), starting in the early morning. Ideally tablets are taken strictly at 6-hourly intervals (6 a.m., 12 noon, 6 p.m., 12 midnight), which may necessitate an inpatient stay. A 24h collection for UFC is taken on the second day of the test, and serum cortisol is measured at 6 a.m. on the third day, 6h after the last dose. IV administration of dexamethasone can be used if there are concerns over absorption or compliance.

•Interpretation: serum cortisol 6h after the last dose should be <140nmol/L, usually <50nmol/L. UFC on the second day should be <70nmol/L, normally <30nmol/L. The 2-day test strictly performed has less false +ves than the overnight test.

•Dexamethasone-suppressed CRH test: dexamethasone 0.5mg is given PO every 6h for nine doses (2 days), but starting at midnight and ending at 6 a.m. Tablets are taken strictly at 6h intervals (12 midnight, 6 a.m., 12 noon, 6 p.m.), which may necessitate an inpatient stay. The last dose is taken at 6 a.m., and an injection of CRH (100µg IV or 1µg/kg) |is given at 8 a.m. A blood sample for cortisol is taken at 8.15 a.m. (i.e. 15min later).

•Interpretation: serum cortisol level should be <38nmol/L (normal).

Further reading

Yanovski JA, Cutler GB Jr, Chrousos GP, Nieman LK. Corticotrophin-releasing hormone stimulation following low-dose dexamethasone administration: a new test to distinguish Cushing’s syndrome from pseudo-Cushing’s states. JAMA 1993; 269: 2232–8.

High-dose dexamethasone suppression test

•Indication: to distinguish between patients with Cushing’s disease (ACTH-secreting pituitary tumour) and ectopic ACTH production in patients with established hypercortisolism.

•Patient preparation: as low-dose test, except that the test can be performed immediately following the 2-day low-dose test.

•Procedure:

1.2 × 24h UFC collections are made to calculate the mean basal 24h UFC.

2.Baseline serum cortisol measurement is also taken before the first dexamethasone dose, ideally at 6 a.m. If the low-dose test is performed first, the baseline values (urine and blood) must be taken prior to the low-dose test (i.e. any doses of dexamethasone).

3.Dexamethasone 2mg is given PO every 6h for eight doses (2 days), starting in the early morning. Ideally tablets are taken strictly at 6h intervals (6 a.m., 12 noon, 6 p.m., 12 midnight), which may necessitate an inpatient stay.

4.A 24h urine collection for UFC (final) is taken on day 2, and a blood sample is taken for (final) cortisol 6h after the last dexamethasone dose (6 a.m. on day 3). Creatinine excretion should be measured and compared between urine samples to confirm true 24h collections.

•Interpretation: the percentage suppression of basal cortisol is calculated as follows:

(Basal cortisol – final cortisol)/basal cortisol × 100

The same calculation is made for basal and day 2 UFC. Fifty per cent suppression is suggestive of pituitary-dependent disease; 90% suppression ↑ the likelihood (strict criteria). Thymic carcinoids and phaeochromocytomas releasing ACTH are sources of false +ves.

Short Synacthen^® test

•Indication: suspected adrenal insufficiency. Will not detect recent-onset 2° adrenal insufficiency.

•Contraindication: asthma/allergy to ACTH—risk of allergic reaction (can be performed with careful medication supervision of the patient).

•Preparation: the patient must not take hydrocortisone on the morning of the test, as this will be detected in the cortisol assay. The test can be performed on low-dose dexamethasone, but the morning dose should be omitted until after the test. May have some value in patients on higher-dose steroid therapy to indicate the degree of suppression of adrenocortical function.

•Procedure: 250µg of synthetic ACTH (Synacthen^®) given IM or IV. Blood taken at times 0, 30, and 60min for serum cortisol.

•Low-dose test: the test can be performed with a very low dose of ACTH (e.g. 1µg). This may detect more subtle degrees of hypoadrenalism, but the clinical significance of these findings remains uncertain.

•Interpretation: a value at any time of >550nmol/L makes the diagnosis very unlikely. (Note: may be lower, depending on local assay range—check with the laboratory).

Long (depot) ACTH test

•Indication: distinguishing 1° and 2° adrenal failure.

•Patient preparation: a short Synacthen^® test should be performed prior to the test to diagnose adrenal failure. If the patient is on steroid replacement, change to dexamethasone 0.5mg/day.

•Procedure: blood is taken at 9 a.m. for basal cortisol; 1mg of depot synthetic ACTH (Synacthen^®) is then given IM on two consecutive days, and blood collected 5h after each dose (2 p.m.). A final cortisol sample is taken at 9 a.m. on the third day.

•Interpretation: serum cortisol should rise to >1000nmol/L on the last day and, if adrenal failure previously indicated by a short Synacthen^® test, such a rise indicates 2° adrenal failure (pituitary/hypothalamic cause, including suppressive drugs).

1 National Institute for Health and Care Excellence. UK NICE guidelines for GH replacement 2003. https://www.nice.org.uk/guidance/ta64.

2 Robertson GL. Diabetes insipidus: diagnosis and management. Best Pract Res Clin Endocrinol Metab 2016; 30: 205–18.

3 Diederich S, Eckmanns T, Exner P, Al-Saadi N, Bähr V, Oelkers W. Differential diagnosis of polyuric/polydipsic syndromes with the aid of urinary vasopressin measurements in adults. Clin Endocrinol (Oxf) 2001; 54: 665–71.

Diabetes mellitus

With normal serum osmolality

With raised osmolality

Primary hyperaldosteronism (decreased renin, increased aldosterone)

Apparent mineralocorticoid excess (decreased renin, increased aldosterone)

Hypercholesterolaemia

Hypertriglyceridaemia