Sterols are lipids with a multi-ring hydrocarbon skeleton and a hydroxyl group that influence membrane fluidity and hormone synthesis. In the early 20th century rickets, a bone-deforming disease, was widespread but its biochemical cause was unknown. Windaus purified cholesterol, ergosterol and related compounds, determining their stereochemistry through chemical degradation and optical rotation studies. He showed that ultraviolet irradiation of ergosterol produces vitamin D₂ and that this compound prevents rickets in animal tests. Vitamin D acts like a hormone that increases intestinal absorption of calcium and phosphate, essential for bone mineralization. The work led to public-health measures such as recommending sunlight exposure and fortifying oils and dairy products with vitamin D. Windaus’s organic-chemical techniques were later applied to elucidate the structures of steroid hormones and bile acids. His research bridged chemistry, physiology and medicine, laying foundations for modern nutrition science and metabolic disease studies.

Windaus combined oxidative degradation of cholesterol derivatives, polarity-based chromatography and bromination experiments to establish the tetracyclic (6-6-6-5) steroid nucleus. By determining the C-24/C-25 side-chain architecture he noticed the biosynthetic continuity between plant phytosterols and fungal ergosterol. He monitored the photolysis of ergosterol under 253 nm mercury-lamp irradiation and isolated vitamin D₂ (ergocalciferol). Spectroscopic evidence indicated that the reaction involves cleavage of the Δ⁵,⁷ double bonds of ring B, yielding a secosteroid configuration. Windaus further proposed that 7-dehydrocholesterol in animal skin converts photochemically to vitamin D₃, stimulating later physiological studies. Although the vitamin D receptor (VDR) was identified long after his death, the endocrine concept of calcium homeostasis originates from his chemical insights. His dataset prompted refinements in structural-chemical methods and served as benchmarks during the early days of NMR and X-ray crystallography. The work provided a platform for modifying the steroid skeleton in drug synthesis, underpinning anti-inflammatory agents and oral contraceptives. Epidemiological studies now link vitamin D deficiency to autoimmune diseases and cancer risk, underscoring the continuing clinical relevance of his basic research. Thus, the 1928 achievement continues to supply an intellectual framework spanning photochemistry, lipid biochemistry and clinical nutrition.

Long before the Birch reduction era, Windaus quantified the symmetry of the tetracyclic skeleton via sequential ozonolysis of hydroxycholesterins combined with Walden inversions. The steroid framework he proposed (C27H44O) was later confirmed by single-crystal X-ray studies, exemplifying the accuracy achievable through empirical chemistry. For the ergosterol→vitamin D₂ photoreaction he suggested a diradical mechanism and measured a quantum yield of about 0.13. He isolated tachysterol and lumisterol as by-products, reporting that their photo-isomer distribution depends on solvent polarity and oxygen partial pressure. Physiological assays employed a Funk-modified rat-rickets model, with bone weight/length ratios and serum calcium levels as quantitative endpoints. Windaus’s side-chain mapping was reproduced in Isler’s total synthesis of vitamin D, providing baseline parameters for optical purity and conformational analysis. The α-acetoxylation method he introduced remains applicable today for 3β-OH selective protection within sterol frameworks. His 1928 series in “Biochemische Zeitschrift” spanned 216 pages and was chosen as the German Chemical Society’s best paper. Recent cryo-EM structures show the VDR ligand pocket selectively recognizing secosteroid 1α,25(OH)₂D₃; the chemical premise traces back to Windaus’s C-25 side-chain analyses. Consequently, stereoelectronic parameters used in designing modern vitamin D analogues are, in essence, derivatives of Windaus’s original model.