Blood Spot LCMS Hormones 7 with SHBG

Testosterone is the principal androgen synthesised by Leydig cells of the testes in males and in smaller amounts by the ovaries and adrenal cortex in females. Circulating testosterone exists in three fractions: tightly bound to sex hormone-binding globulin (approximately 60%), loosely bound to albumin (approximately 38%), and free (1–2%). Liquid chromatography–tandem mass spectrometry measures total testosterone with superior specificity and sensitivity at low concentrations compared with immunoassay, making it the gold standard for females, children, and hypogonadal males.

Testosterone measurement is central to the evaluation of hypogonadism in males, hyperandrogenism in females, and disorders of sexual development. In males low testosterone prompts assessment of hypothalamic–pituitary–testicular axis integrity; in females elevated testosterone suggests polycystic ovary syndrome, congenital adrenal hyperplasia, or androgen-secreting tumours. The use of LC-MS on a dried blood spot enables convenient at-home collection while retaining the analytical rigour required for low-concentration samples.

Oestradiol is the most potent naturally occurring oestrogen, synthesised by granulosa cells of the ovaries in females and through peripheral aromatisation of testosterone in males. It regulates reproductive cycling, bone density, lipid metabolism, and cardiovascular function. LC-MS measurement avoids the cross-reactivity and poor sensitivity at low concentrations that plague immunoassays, making it essential for accurate assessment in males, postmenopausal females, and children.

In females oestradiol measurement aids investigation of amenorrhoea, menstrual irregularity, infertility, and menopausal status; in males elevated oestradiol may signal obesity-driven aromatisation or oestrogen-secreting tumours, while very low levels can impair bone health. The ratio of oestradiol to testosterone is increasingly recognised as relevant to metabolic and cardiovascular risk in both sexes.

Progesterone is secreted by the corpus luteum after ovulation and by the placenta during pregnancy. In the luteal phase of the menstrual cycle it prepares the endometrium for implantation and modulates immune and inflammatory pathways. Small amounts are synthesised by the adrenal cortex in both sexes and serve as a precursor for other steroid hormones.

Mid-luteal progesterone measurement confirms ovulation and adequate corpus luteum function. Low luteal values suggest anovulation or luteal-phase deficiency, a contributor to infertility and early pregnancy loss. Elevated progesterone outside the luteal phase or pregnancy may indicate adrenal hyperplasia or an ovarian tumour.

DHEA is an androgen precursor secreted primarily by the zona reticularis of the adrenal cortex. It circulates predominantly as the sulphate ester DHEA-S, which serves as a reservoir; free DHEA is the unconjugated, biologically active form. DHEA is further metabolised to androstenedione and testosterone peripherally. Its secretion peaks in early adulthood and declines with age, a phenomenon termed adrenopause.

DHEA measurement assists in distinguishing adrenal from gonadal sources of androgen excess and in assessing adrenal reserve. Elevated DHEA points to adrenal hyperplasia or tumours; low DHEA may indicate adrenal insufficiency, hypopituitarism, or age-related decline. DHEA is also used off-label as a supplement, so interpretation must account for exogenous intake.

Cortisol is the principal glucocorticoid, synthesised in the zona fasciculata of the adrenal cortex under ACTH control. It follows a diurnal rhythm, peaking in the early morning and reaching a nadir around midnight. Cortisol regulates glucose metabolism, immune function, vascular tone, and the stress response. Approximately 90% circulates bound to cortisol-binding globulin and albumin; the free fraction is biologically active.

Cortisol measurement is central to the diagnosis of Cushing syndrome (elevated) and adrenal insufficiency (low). Single time-point values are less useful than paired serum cortisol with ACTH, late-night salivary cortisol, or 24-hour urinary free cortisol, but a dried blood spot offers a convenient screening option when logistics or patient preference preclude venepuncture.

Cortisone is the inactive 11-keto metabolite of cortisol, formed by 11β-hydroxysteroid dehydrogenase type 2 in the kidney and other tissues. It serves as a reservoir that can be reconverted to cortisol by the type 1 isoenzyme. Measurement of cortisone alongside cortisol can provide insight into the activity of these interconverting enzymes and has been proposed as a less-variable marker of hypothalamic–pituitary–adrenal axis activity, though its clinical utility remains under investigation.

Cortisone measurement is not routine in standard endocrine assessment. Some functional-medicine practitioners interpret the cortisol-to-cortisone ratio as an index of 11β-HSD activity and tissue glucocorticoid metabolism, but this approach is not widely adopted in mainstream practice.

The testosterone-to-progesterone ratio is a calculated index that reflects the balance between androgenic and progestogenic activity. In females a high T/P4 ratio during the luteal phase has been proposed as a marker of relative androgen excess in the setting of luteal insufficiency, a pattern sometimes seen in polycystic ovary syndrome.

The T/P4 ratio is not a standard diagnostic criterion but is used by some practitioners as an adjunct to traditional measures of androgen excess and ovulatory function. It should be interpreted alongside absolute testosterone, progesterone, LH, and clinical features rather than in isolation.

SHBG is a glycoprotein synthesised by the liver that binds testosterone and oestradiol with high affinity, thereby regulating the bioavailable fraction of these hormones. Factors that increase SHBG (thyroid hormone, oestrogen, ageing, low BMI) reduce free androgen availability; factors that decrease it (insulin resistance, obesity, androgens, hypothyroidism) increase free hormone availability.

SHBG measurement allows calculation of free or bioavailable testosterone when direct free-testosterone assays are unavailable or unreliable. Low SHBG is a marker of insulin resistance and metabolic syndrome; high SHBG may mask true androgen deficiency by lowering the free fraction despite normal total testosterone. SHBG is essential to the interpretation of total testosterone in both sexes.