25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3

Cholangiocarcinoma (CCA) is a devastating disease without effective treatments. 1α,25(OH)₂D₃, the active form of Vitamin D, has emerged as a new anti-cancer regimen. However, the side effect of hypercalcemia impedes its systemic administration. 25(OH)D is biologically inert and needs hydroxylation by CYP27B1 to form 1α,25(OH)₂D₃, which is originally believed to only take place in kidneys. Recently, the extra-renal expression of CYP27B1 has been identified and in vitro conversion of 25(OH)D to 1α,25(OH)₂D₃ has been found in some cancer cells with CYP27B1 expression. In this study, CYP27B1 expression was demonstrated in CCA cells and human CCA specimens. 25(OH)D effectively represses SNU308 cells growth, which was strengthened or attenuated as CYP27B1 overexpression or knockdown. Lipocalcin-2 (LCN2) was also found to be repressed by 25(OH)D. After treatment with 800 ng/mL 25(OH)D, the intracellular 1α,25(OH)₂D₃ concentration was higher in SNU308 cells with CYP27B1 overexpression than wild type SNU308 cells. In a xenograft animal experiment, 25(OH)D, at a dose of 6 μg/kg or 20 μg/kg, significantly inhibited SNU308 cells’ growth without inducing obvious side effects. Collectively, our results indicated that SNU308 cells were able to convert 25(OH)D to 1α,25(OH)₂D₃ and 25(OH)D CYP27B1 gene therapy could be deemed as a promising therapeutic direction for CCA.     

Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism

Two 24-fluoro-25-hydroxyvitamin D₃ analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D₃ side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D₃ revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D₃ (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.     

Investigation of Vitamin D2 and Vitamin D3 Hydroxylation by Kutzneria albida

The active vitamin D metabolites 25-OH−D and 1α,25-(OH)₂−D play an essential role in controlling several cellular processes in the human body and are potentially effective in the treatment of several diseases, such as autoimmune diseases, cardiovascular diseases and cancer. The microbial synthesis of vitamin D₂ (VD₂) and vitamin D₃ (VD₃) metabolites has emerged as a suitable alternative to established complex chemical syntheses. In this study, a novel strain, Kutzneria albida, with the ability to form 25-OH−D₂ and 25-OH−D₃ was identified. To further improve the conversion of the poorly soluble substrates, several solubilizers were tested. 100-fold higher product concentrations of 25-OH−D₃ and tenfold higher concentrations of 25-OH−D₂ after addition of 5 % (w/v) 2-hydroxypropyl β-cyclodextrin (2-HPβCD) were reached. Besides the single-hydroxylation products, the human double-hydroxylation products 1,25-(OH)₂−D₂ and 1,25-(OH)₂−D₃ and various other potential single- and double-hydroxylation products were detected. Thus, K. albida represents a promising strain for the biotechnological production of VD₂ and VD₃ metabolites.     

Two Major Bile Acids in the Hornbills, (24R,25S)-3α,7α,24-Trihydroxy-5β-cholestan-27-oyl Taurine and Its 12α-Hydroxy Derivative

Two major bile acids were isolated from the gallbladder bile of two hornbill species from the Bucerotidae family of the avian order Bucerotiformes Buceros bicornis (great hornbill) and Penelopides panini (Visayan tarictic hornbill). Their structures were determined to be 3α,7α,24‐dihydroxy‐5β‐cholestan‐27‐oic acid and its 12α‐hydroxy derivative, 3α,7α,12α,24‐tetrahydroxy‐5β‐cholestan‐27‐oic acid (varanic acid, VA), both present in bile as their corresponding taurine amidates. The four diastereomers of varanic acid were synthesized and their assigned structures were confirmed by X‐ray crystallographic analysis. VA and its 12‐deoxy derivative were found to have a (24R,25S)‐configuration. 13 additional hornbill species were also analyzed by HPLC and showed similar bile acid patterns to B. bicornis and P. panini. The previous stereochemical assignment for (24R,25S)‐VA isolated from the bile of varanid lizards and the Gila monster should now be revised to the (24S,25S)‐configuration.     

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells

Background: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25D₃, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. Methods: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D₃ 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). Results: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC₅₀ = 2.98 ± 1.10 nmol/L, in 14433 cells: EC₅₀ = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC₅₀ = n/d, in 14433 cells: EC₅₀ = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. Conclusions: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs’ structure might lead to new treatment options against ovarian cancer.     

New cis-configured aziridine-2-carboxylates as aspartic acid protease inhibitors

A series of 52 cis‐configured 1‐alkyl‐3‐phenylaziridine‐2‐carboxylates were synthesized as new pseudo‐irreversible inhibitors of Candida albicans secreted aspartic acid protease 1 (SAP1), SAP2, SAP3, and SAP8. Some of the compounds, which were obtained as diastereomers with S,S‐ and R,R‐configured aziridine rings by Cromwell synthesis of racemic (2R,3S+2S,3R)‐dibromophenylpropionic acid ester with amines, followed by ester hydrolysis and coupling to hydrophobic amino acid esters, were separated by preparative HPLC. The absolute configuration of the aziridine ring was assigned by a combination of experimental circular dichroism (CD) investigations and quantum chemical CD calculations. In agreement with previous docking studies, the diastereomers all exhibit similar activity. The compounds were found to be more active against the related mammalian enzyme cathepsin D, presumably due to productive interactions of the N‐alkyl substituent with the highly lipophilic S2 pocket. The most active inhibitors ( 5 , 9 , 10 , 21 , and 28 ), characterized by benzyl, cyclohexylmethyl, tert‐butyl, or 1,4‐dimethylpentyl moieties at the aziridine nitrogen atom, exhibit k_2nd values between 500 and 900×10³ M ^?1 min^?1 and K_i values near or below 1 μM for cathepsin D.     

Substrate Enantioselective and Product Stereoselective Metabolism of Racemic 2-Hydroxy-3-methylcholanthrene by Rat Liver Microsomes

Racemic 2-hydroxy-3-methylcholanthrene (rac-2-OH-3MC) is a potent carcinogen and 2S-OH-3MC is the most abundant metabolite formed in the metabolism of 3-methylcholanthrene (3MC) by rat liver microsomes. Eighteen identifiable metabolites were formed in the metabolism of rac-2-OH-3MC by liver microsomes prepared from phenobarbital-treated rats. Metabolites were separated by sequential use of reversed-phase and normal-phase high performance liquid chromatography (HPLC) and characterized by ultraviolet-visible absorption, mass, and circular dichroism spectral, and chiral stationary phase (CSP) HPLC analyses. Identified metabolites were: 2-OH-3MC 7,8-dihydrodiol (enriched in 7R,8R enantiomer), 2-OH-3MC 9,10-dihydrodiol (2S,9R,10R:2R,9S,10S ≈ 27:73 and 2R,9R,10R:2S,9S,10S ≈ 81:19), 2-OH-3MC 11,12-dihydrodiol (2R,11R,12R:2S,11S,12S ≈ 5:95 and an essentially optically pure 2S,11R,12R enantiomer), 3MC trans-1,2-diol (1S,2S:1R,2R ≈ 68:32), 3MC cis-1,2-diol (1S,2R:1R,2S ≈ 18:82), 2-OH-3-hydroxymethyl-cholanthrene (2-OH-3-OHMC, 2R:2S ≈ 19:81), 2-OH-3-OHMC 9,10-dihydrodiol (9R,10R:9S,10S ≈ 62:38), 3MC-2-one 9,10-dihydrodiol (9R,10R:9S,10S ≈ 7:3), 3MC cis-1,2-diol:trans-9,10-dihydrodiol, 8-hydroxy-2-OH-3-OHMC, 9-hydroxy-2-OH-3MC, 10-hydroxy-2-OH-3MC, and 3MC-2-one. The enantiomer ratios of some metabolites formed were determined by circular dichroism spectra and/or CSP-HPLC. Six 9,10-dihydrodiols formed in the metabolism of 2-OH-3MC may be further converted to potentially reactive 9,10-diol-7,8-epoxides and may contribute to the overall carcinogenicity exhibited by 3MC.     

CYP24A1 Expression Inversely Correlates with Melanoma Progression: Clinic-Pathological Studies

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3) homeostasis. Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities. Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas. Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases. In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis. The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas. With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases. Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity. Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes. Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival. In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.     

A Single Mutation at the Ferredoxin Binding Site of P450 Vdh Enables Efficient Biocatalytic Production of 25‐Hydroxyvitamin D3

Vitamin D₃ hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two‐step hydroxylation of vitamin D₃ (VD₃) to produce 25‐hydroxyvitamin D₃ (25(OH)VD₃) and 1α,25‐dihydroxyvitamin D₃ (1α,25(OH)₂VD₃). These hydroxylated forms of VD₃ are useful as pharmaceuticals for the treatment of conditions associated with VD₃ deficiency and VD₃ metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin‐binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD₃, a promising intermediate for the synthesis of various hydroxylated VD₃ derivatives, by using nisin‐treated Rhodococcus erythropolis cells containing Vdh_T107A. The gene‐expression cassette encoding Bacillus megaterium glucose dehydrogenase‐IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL^?1 25(OH)VD₃ was successfully produced by a 2 h bioconversion.     

Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D2 as Potential Anti-Leukemic Agents

Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D₂ (1,25D₂) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D₂. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D₂ and 1,25D₃, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D₂. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist.     

Anomalous enantioselectivity in the sharpless asymmetric dihydroxylation reaction of 24-nor-5β-cholest-23-ene-3α,7α,12α-triol: Synthesis of substrates for studies of cholesterol side-chain oxidation

Recently we described a block in bile acid synthesis in cerebrotendinous xanthomatosis (CTX), a lipid storage disease related to an inborn error of bile acid metabolism. In this disease a defect in hepatic microsomal (24S) hydroxylation blocks the transformation of 5β-cholestane-3α,7α,12α,25-tetrol into (24S) 5β-cholestane-3α,7α,12α,24,25-pentol and cholic acid. Mitochondrial cholesterol 27-hydroxylation has also been reported to be abnormal in CTX subjects, but the relative importance of the enzymatic defect in this alternative microsomal pathway (namely, the 24S hydroxylation of 5β-cholestane-3α,7α,12α,25-tetrol relative to the abnormality in mitochondrial 27-hydroxylase) has not been established in CTX. To delineate the sequence of side-chain hydroxylations and the enzymatic block in bile acid synthesis, we synthesized the (23 R and 23 S) 24-nor-5β-cholestane-3α,7α,12α,23,25-pentols utilizing a modified Sharpless asymmetric dihydroxylation reaction on 24-nor-5β-cholest-23-ene-3α,7α,12α-triol, a C₂₆ analog of the naturally occurring C₂₇ bile alcohol, 5β-cholest-24-ene-3α,7α,12α-triol. Stereospecific conversion of the unsaturated 24-nor triol to the corresponding chiral compounds (23R and 23S), 24-nor-5β-cholestane-3α,7α,12α,23,25-pentols, was quantitative. However, conversion of the unsaturated 24-nor triol to the chiral nor-pentols had absolute stereochemistry opposite to the products predicted by the Sharpless steric model. The absolute configurations and enantiomeric excess of the C₂₆ nor-pentols and the C₂₇ pentols (synthesized from 5β-cholest-24-ene-3α,7α,12α-triol for comparison) were confirmed by nuclear magnetic resonance and lanthanide-induced circular dichroism Cotton effect measurements. These results may contribute to a better understanding of the role of the 24S-hydroxylation vs. 27-hydroxylation step in cholic acid biosynthesis. Presented in part at the 216th American Chemical Society National Meeting (Medicinal Chemistry Division, Abs. 368), Boston, MA, August 21–26, 1998.     

Recent Developments of 19‐Nor‐1,25‐dihydroxyvitamin D3 Analogues

The vitamin D hormone, 1α,25‐dihydroxyvitamin D₃ [1,25‐(OH)₂D₃], exerts its hormonal effects predominantly on intestine, bone, and kidney, where it plays a crucial role in calcium and phosphorus homeostasis and bone mineralization. In addition to its classical actions, 1,25(OH)₂D₃ exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)₂D₃ have suggested a multitude of potential therapeutic applications for the vitamin D hormone in the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). However, the calcemic effects induced by 1,25(OH)₂D₃—hypercalcemia, increased bone resorption, and soft tissue calcification—limit the use of the natural ligand in these clinical applications. Therefore, numerous 1,25(OH)₂D₃ analogues have been synthesized with the intent of producing therapeutic agents devoid of hypercalcemic and hyperphosphatemic side effects. To this aim, much attention has been focused on the development of 19‐nor‐vitamin D₃ derivatives that lack the ring‐A exocyclic methylene group (C19). In this review, the 19‐nor‐1,25(OH)₂D₃ analogues are classified according to modifications made at the A‐ring, the side chain, or both the A‐ring and side chain, as well as other positions. The biological activities of these 19‐nor‐1,25(OH)₂D₃ analogues are summarized and their structure–activity relationships and binding features with the vitamin D receptor (VDR) are discussed.     

Antitumor Effects of Vitamin D Analogs on Hamster and Mouse Melanoma Cell Lines in Relation to Melanin Pigmentation

Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)₂D₃) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)₂D₃ and 25(OH)D₃), and novel relatively non-calcemic ones (20(OH)D₃, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner.     

Mechanistic Insights into the Cytochrome P450‐Mediated Oxidation and Rearrangement of Littorine in Tropane Alkaloid Biosynthesis

During the biosynthesis of certain tropane alkaloids, littorine ( 1 ) is rearranged to hyoscyamine ( 3 ). Recent evidence indicates that this isomerisation is a two‐step process in which the first step is an oxidation/rearrangement to give hyoscyamine aldehyde ( 2 ). This step is catalysed by CYP80F1, a cytochrome P450 enzyme, which was recently identified from the plant Hyoscyamus niger; CYP80F1 also catalyses the hydroxylation of littorine at the 3′‐position. The mechanisms of the reactions catalysed by CYP80F1 were probed with synthetic deutero and arylfluoro analogues of 1 . Measurement of the primary kinetic isotope effects indicates that C3′ hydrogen abstraction is the rate‐limiting step for the oxidation/rearrangement of natural littorine, and for the 3′‐hydroxylation reaction of the unnatural S enantiomer of littorine. The character of the intermediates in the oxidation/rearrangement and hydroxylation reaction was probed with the use of arylfluorinated analogues of (R)‐littorine (natural stereoisomer) and (S)‐littorine (unnatural stereoisomer) as substrates for CYP80F1. The relative conversions of ortho‐, meta‐ and para‐fluorolittorine analogues were used to obtain information on the likely intermediacy of either a benzylic radical or benzylic carbocation intermediate. The data suggest that hydroxylation takes place via a benzylic carbocation intermediate, whereas the product profile arising from rearrangement is more consistent with a benzylic radical intermediate.     

Carving the Active Site of CYP153A7 Monooxygenase for Improving Terminal Hydroxylation of Medium-Chain Fatty Acids

The P450-mediated terminal hydroxylation of non-activated C−H bonds is a chemically challenging reaction. CYP153A7 monooxygenase, discovered in Sphingomonas sp. HXN200, belongs to the CYP153A subfamily and shows a pronounced terminal selectivity. Herein, we report the significantly improved terminal hydroxylation activity of CYP153A7 by redesign of the substrate binding pocket based on molecular docking of CYP153A7−C_8:0 and sequence alignments. Some of the resultant single mutants were advantageous over the wild-type enzyme with higher reaction rates, achieving a complete conversion of n-octanoic acid (C_8:0, 1 mM) in a shorter time period. Especially, a single-mutation variant, D258E, showed 3.8-fold higher catalytic efficiency than the wild type toward the terminal hydroxylation of medium-chain fatty acid C_8:0 to the high value-added product 8-hydroxyoctanoic acid.     

Synthesis and Pharmacological Evaluation of Enantiomerically Pure GluN2B Selective NMDA Receptor Antagonists

To determine the eutomers of potent GluN2B‐selective N‐methyl‐d ‐aspartate (NMDA) receptor antagonists with a 3‐benzazepine scaffold, 7‐benzyloxy‐3‐(4‐phenylbutyl)‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ols (S)‐ 2 and (R)‐ 2 were separated by chiral HPLC. Hydrogenolysis and subsequent methylation of the enantiomerically pure benzyl ethers of (S)‐ 2 and (R)‐ 2 provided the enantiomeric phenols (S)‐ 3 and (R)‐ 3 [3‐(4‐phenylbutyl)‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine‐1,7‐diol] and methyl ethers (S)‐ 4 and (R)‐ 4 . All enantiomers were obtained with high enantiomeric purity (≥99.7 % ee). The absolute configurations were determined by CD spectroscopy. R‐configured enantiomers turned out to be the eutomers in receptor binding studies and two‐electrode voltage clamp experiments. The most promising ligand of this compound series is the R‐configured phenol (R)‐ 3 , displaying high GluN2B affinity (K_i=30 nm ), high inhibition of ion flux (IC₅₀=61 nm ), and high cytoprotective activity (IC₅₀=93 nm ). Whereas the eudismic ratio in the receptor binding assay is 25, the eudismic ratio in the electrophysiological experiment is 3.     

GC/EI-MS Determination of the Diastereomer Distribution of Phytanic Acid in Food Samples

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid, produced by bacteria by means of oxidation and biohydrogenation of the chlorophyll side chain phytol (3,7R,11R,15-tetramethylhexadec-2-en-1-ol). The later reaction generates to a new stereogenic center on C-3 which can be both 3R- or 3S-configured. Thus, two diastereomers (3S,7R,11R,15- and 3R,7R,11R,15-phytanic acid) are naturally produced. In this study we examined the diastereomer composition of phytanic acid in terrestrial and marine food samples. Phytanic acid was transferred into its methyl ester which was analyzed by GC/MS in the selected ion monitoring mode. The first eluted diastereomer in the samples was tentatively identified as 3S,7R,11R,15-phytanic acid. The marine samples were clearly dominated by 3S,7R,11R,15-phytanic acid whose abundance was higher in marine mammals than in fish. Milk from one organic cow collected over a period of 30 days showed lower proportions of 3S,7R,11R,15-phytanic acid than milk from one cow raised with conventional feed. The difference between organic and conventional dairy products (cheese and butter) was not as pronounced as in milk. Milk samples from other mammals (goat, sheep, mare, camel, moose, and human) also showed an excess of 3S,7R,11R,15-phytanic acid except for camel and moose milk.