DUTCH Plus (Dried Urine Test for Comprehensive Hormones, Plus Salivary Cortisol Awakening Response)

Cortisol is the principal glucocorticoid, secreted by the adrenal cortex under hypothalamic-pituitary (CRH-ACTH) control with a marked diurnal rhythm peaking after waking and declining to a nocturnal nadir. The free, unbound fraction is the biologically active portion and is what dried-urine collection across the day aims to capture as a circadian pattern. Free cortisol reflects the bioavailable hormone rather than the protein-bound majority measured by total serum cortisol.

The diurnal free cortisol pattern is used in functional and integrative practice to characterise the shape of the daily cortisol curve — its waking value, slope, and nocturnal level — rather than a single point. It is interpreted to assess HPA-axis function and patterns attributed to chronic stress. It does not replace serum or salivary cortisol or dynamic testing (such as the dexamethasone suppression or ACTH stimulation tests) for diagnosing Cushing's syndrome or adrenal insufficiency.

The cortisol awakening response is the sharp rise in cortisol occurring within roughly 30–45 minutes of waking, superimposed on the diurnal rhythm. It is captured through timed salivary samples at waking and shortly after, and reflects the reactivity of the HPA axis to the transition from sleep to wakefulness. The salivary route measures free cortisol. This salivary CAR is the component that distinguishes DUTCH Plus from the standard DUTCH Complete.

The CAR is used as an index of HPA-axis reactivity, with a blunted or exaggerated response interpreted in the context of chronic stress, fatigue, and circadian disruption in functional practice. Its measurement is sensitive to collection timing and adherence, and it is a research and functional-assessment tool rather than an established standalone diagnostic for defined endocrine disease.

Cortisol is metabolised by hepatic and peripheral enzymes (including 5-alpha and 5-beta reductases) to tetrahydro metabolites excreted in urine. The sum of these metabolites estimates total cortisol production, which can differ from free cortisol levels when clearance or peripheral metabolism is altered, for example in obesity or thyroid dysfunction. Measuring both free and metabolised cortisol aims to distinguish production from clearance effects.

Metabolised cortisol provides an estimate of overall cortisol output that complements the free cortisol pattern; a discordance between low free cortisol and high metabolised cortisol may suggest rapid clearance rather than low production. This is used interpretively in functional assessment and is not a substitute for established dynamic adrenal testing.

DHEA-S is the principal adrenal androgen precursor, while downstream androgens including testosterone are metabolised via 5-alpha and 5-beta pathways to androsterone and etiocholanolone, excreted in urine. The relative activity of 5-alpha reductase, inferred from metabolite ratios, influences potent androgen exposure at tissue level. Dried urine captures these metabolites to profile androgen production and metabolism.

Androgen and DHEA-S metabolites are used to assess adrenal androgen output and androgen metabolism in conditions such as PCOS, androgenic symptoms, and adrenal function assessment. The 5-alpha/5-beta metabolite balance is interpreted to gauge androgen activity. Findings inform functional assessment and are interpreted alongside serum measurements where definitive endocrine diagnosis is required.

Oestrogens (oestrone, oestradiol, oestriol) are metabolised through three hydroxylation pathways — 2-hydroxy, 4-hydroxy, and 16-hydroxy — with subsequent methylation. The relative flux through these pathways and the degree of methylation are of interest because the 4-OH pathway generates more reactive metabolites. Dried urine measures parent oestrogens and these downstream metabolites together.

Oestrogen metabolite profiling is used in functional practice to examine oestrogen production and the balance of metabolic pathways, including methylation capacity. Interpretation regarding pathway 'preference' and associated risk is an area of active research and is not an established clinical risk-stratification tool. It is interpreted with cycle timing, menopausal status, and any hormone therapy.

Progesterone is metabolised to pregnanediol isomers excreted in urine, which serve as a surrogate for progesterone production over the collection window. Because progesterone itself is not measured directly in dried urine, these metabolites are used to estimate luteal-phase progesterone activity. Levels vary markedly with cycle phase and are minimal outside the luteal phase and in postmenopausal women.

Progesterone metabolites are used to estimate luteal-phase progesterone production and to assess the oestrogen-to-progesterone balance in functional hormone assessment. As a urinary surrogate, the result reflects metabolism over the collection period rather than a single serum value, and cycle timing is essential for interpretation.

Melatonin, secreted by the pineal gland with a nocturnal peak, is metabolised principally to 6-hydroxymelatonin sulphate, which is excreted in urine and serves as a validated surrogate of melatonin production. The dried-urine waking sample captures overnight melatonin output. It reflects circadian melatonin secretion rather than an instantaneous level.

Urinary 6-OHMS is used to estimate overnight melatonin production in the assessment of circadian rhythm and sleep complaints within functional practice. It complements the cortisol rhythm in characterising circadian function and is interpreted as a surrogate of melatonin output.

8-Hydroxy-2-deoxyguanosine (8-OHdG) is a urinary marker of oxidative DNA damage. Selected organic acid markers reported alongside the hormone panel reflect aspects of neurotransmitter metabolism (such as dopamine and noradrenaline metabolites) and B-vitamin and glutathione-related pathways. These are measured from the same dried-urine specimen as adjunctive metabolic markers.

8-OHdG is used as an indicator of systemic oxidative stress, and the organic acid markers are used adjunctively to flag possible nutrient or neurotransmitter-metabolism patterns for further evaluation. The clinical interpretation of several of these markers is not firmly established, so they are treated as supportive context rather than diagnostic findings.