Quantitation of serum 25(OH)D2 and 25(OH)D3 concentrations by liquid chromatography tandem mass spectrometry in patients with diabetes mellitus

Vitamin D has been considered to regulate calcium and phosphorus homeostasis and to preserve skeletal integrity. Serum 25-hydroxyvitamin D (25(OH)D) is the best indicator of vitamin D levels. The association of serum 25(OH)D deficiency with increased risk of type 1 diabetes (T1DM) and type 2 diabetes (T2DM) is controversial. We investigated serum 25(OH)D₂ and 25(OH)D₃ levels in diabetes patients by using liquid chromatography tandem mass spectrometry (LC-MS/MS). Serum 25(OH)D2 and 25(OH)D3 levels were measured with liquid chromatography tandem mass spectrometry in electrospray ionization positive mode. Chromatograms were separated using an ACE5 C18 column on a gradient of methanol. The total 25(OH)D levels were calculated as the sum of 25(OH)D3 and 25(OH)D2 levels. A total of 56 patients with T1DM and 41 patients with T2DM were enrolled in this study. There were 42 and 28 non-diabetic, age-matched volunteers who participated as the T1DM controls and the T2DM controls, respectively. The total 25(OH)D levels were lowest in the 21–40 age group. The levels of both 25(OH)D3 and the total 25(OH)D were significantly higher in the T1DM and T2DM groups than in the controls (p < 0.01 in T1DM and p < 0.05 in T2DM group, respectively). The 25(OH)D2 levels were only significantly higher in T1DM patients than in the controls. The percentages of vitamin D deficiency (total 25(OH)D less than 20 ng/mL) in the T1DM, T2DM, the T1DM controls and the T2DM controls were 7.1%, 0%, 14.3% and 3.6%, respectively. The percentages of vitamin D insufficiency (total 25(OH)D less than 30 ng/mL) in the T1DM, T2DM, the T1DM controls and the T2DM controls were 26.8%, 7.3%, 54.8% and 17.9%, respectively. The percentages of vitamin D deficiency and insufficiency were significantly lower in the T1DM patients than in the T1DM controls (p < 0.01). In the present study, both type 1 and type 2 diabetes patients had higher serum 25(OH)D levels and lower percentages of vitamin D deficiency/insufficiency.     

孕期肥胖与25-OH维生素D的关系

目的探讨孕期体重指数（BMI）对孕妇体内25-OH维生素D含量的影响。方法选取作产前检查的398例单胎初产妇,根据体重指数（BMI）分为三组,体质量正常组（BMI〈25kg／m^2）;超重组（25〈BMI〈29．9kg／m^2）;肥胖组（BMI〉30kg／m^2）。采用放免法分别在4—22孕周和产前（37～41孕周）测定孕妇体内25．OH维生素D的含量,并校正孕妇年龄、孕周、维生素使用情况、基础血压等之后,应用Spearman等级相关系数评估孕妇体重指数（BMI）与25-OH维生素D的相关性。结果肥胖组体内25-OH维生素D含量显著低于体重正常组（P〈0．05）;孕妇BMI与血清25-OH维生素D在4—21孕周（r=-0．22,P〈0．001）和产前（r=0．19,P〈0．001）呈显著负相关。结论孕期25-OH维生素D含量下降与孕妇肥胖有关。     

25-(OH)D3与相关血液微量元素的相关性研究

目的 测定不同年龄段儿童的血清25-(OH)D3(25-羟维生素D3)的含量,并探究25-(OH)D3与BAKP(骨碱性磷酸酶)及微量元素的相关性.方法 随机选择382例本院儿童保健所门诊正常体检儿童作为研究对象,根据年龄分为0～1岁、1～2岁、2～3岁、3～4岁、4～5岁共5组,采用酶联免疫测试法检测血清25-(OH) D3含量,并测定血清BAKP及Cu、Zn、Fe、Mg等微量元素含量,分析25-(OH) D3与BAKP、微量元素的相关性.结果 各年龄段儿童均明显存在25-(OH) D3缺乏的现象.采用Pearson相关性检验表明,Zn和BAKP、Cu,Fe和BAKP、Cu、Zn,Mg和Cu、Z、Fe之间均存在明显的相关性(P＜0.05);25-(OH) D3与BAKP存在相关性(P＜0.05);而25-(OH) D3与Cu、Zn、Fe、Mg等微量元素之间均无明显相关性(P＞0.05).结论 0～5岁的儿童均不同程度地缺乏25-(OH) D3,尤其是0～2岁的儿童缺乏25-(OH) D3现象严重.相关性分析说明25-(OH) D3与BAKP存在相关性,与Cu、Zn、Fe、Mg等微量元素无相关性,补充Zn和Fe可有助于改善BAKP含量,对改善儿童身体健康具有重要意义.     

1,25 (OH)2 vitamin D3 levels in seasonal affective disorder: Effects of light

1,25 (OH)₂ vitamin D₃ levels were assessed in nine females and six males with winter seasonal affective disorder and in 15 age- and gender-matched controls in winter during periods with and without treatment with light therapy. No difference was found between groups or across conditions, though wide variations within each group make the presence of a type II error possible.     

1,25(OH)2D3在牙乳头干细胞成骨分化期的作用研究

摘要 目的：研究1,25(OH)2D3在牙乳头干细胞向成骨细胞分化过程中的作用角色。方法：分离培养牙乳头干细胞,培养基中添加不同浓度1,25(OH)2D3,MTT法检测细胞生长速度,流式细胞术检测细胞增殖周期变化,western blot方法检测核因子-k B受体活化剂配体（RANKL）、骨保护素（OPG）和维生素D受体（VDR）的蛋白质表达水平;siRNA技术沉默VDR表达后,检测其对RANKL和OPG蛋白质表达的影响。结果：MTT和流式细胞术检测结果显示1,25(OH)2D3对牙乳头干细胞的增殖没有明显作用;western blot结果显示RANKL、OPG和VDR的蛋白质表达与1,25(OH)2D3浓度具有剂量相关性;siRNA沉默VDR表达后,RANKL和OPG的蛋白质表达水平均有不同程度下降。结论：1,25(OH)2D3通过调节VDR水平影响牙乳头干细胞向成骨细胞方向的分化过程。     

Using gas chromatography and mass spectrometry to determine 25-hydroxyvitamin D levels for clinical assessment of vitamin D deficiency

Vitamin D is responsible for multiple metabolic functions in humans. Rickets are the most common disease caused by vitamin D deficiency. It is caused by poor calcium intake resulting in poor serum-ionized calcium. The purpose of this study is to develop a rapid, sensitive, and feasible method to determine the 25-hydroxy-vitamin D3 (25(OH)D3) levels in blood samples for clinical assessment. In this study, gas chromatography coupled mass spectrometry with trimethylsilyl derivatization (TMS-GC-MS) is the most suitable protocol for quantitative analyses of 25(OH)D3. Performance of method was evaluated and compared with liquid chromatography and immunoassay. Method validation has been carried out with plasma specimens. The limit of quantitation of TMS-GC-MS method is 1.5 ppb with good linear correlation. Furthermore, the dietary intake and nutritional status of vegetarian and non-vegetarians in Taiwan were assessed by our validated method. As a result, this vitamin D nutrition survey demonstrates that most Taiwanese people have insufficient vitamin D. Due to dietary habits; the male vegans may have the highest risk of vitamin D deficiency.     

Hypercalcemia,hypervitaminosis A and 3-epi-25-OH-D3 levels after consumption of an “over the counter” vitamin D remedy. A case report

Intoxication from vitamin D supplements has been rarely reported but, nowadays, it occurs more frequently. 3-epi-25-OH-D₃ is highly prevalent in adults and it is considered of biological relevance. We report a case of vitamin D toxicity with hypercalcemia, acute renal failure and hypervitaminosis A after consuming an over-the-counter vitamin D supplement. Our data suggest that the contribution of 3-epi-25-OH-D₃ is not altered during vitamin D toxicity, although the serum levels of 25-OH-D₃ and 3-epi-25-OH-D₃ may display a different rate of clearance. The patient also displayed hypervitaminosis A unrelated to diet, possibly caused by renal failure related to the hypercalcemia induced by vitamin D toxicity. Because of the increasing use of over-the-counter vitamin D supplements and the potential iatrogenic hypercalcemia related to hypervitaminosis A, the present case highlights the importance of evaluating both the use of (non-) prescribed medication and vitamin A status during vitamin D toxicity.     

呋苄头孢菌素和呋苄青霉素钾的极谱行为研究

本文研究呋苄头孢菌素和呋苄青霉素钾的极谱行为。两样品在-1.1 V附近有一清晰的单扫描极谱波,在一定的浓度范围内蜂电流与样品浓度之间呈良好的线性关系,可用作定量分析。本文还对电极反应机理进行了初步的探讨,并证明此波具有吸附性质。     

哮喘儿童血清25-（OH）D3、总IgE、特异性IgE的相关性研究

目的：研究支气管哮喘儿童血清25-羟胆骨化醇[25-(OH)D3]、总免疫球蛋白E(TIgE)及特异性IgE（sIgE）的变化及临床意义。方法将60例健康体检儿童为对照组,180例确诊为支气管哮喘的患儿为哮喘组,哮喘组根据年龄分为婴幼儿组（<5岁）60例及儿童组（≥5岁）120例,分别进行过敏原特异性IgE（采用免疫印迹法）、血清总IgE,血清25-(OH)D3的检测及肺功能（5岁以上）及气道阻力测定（5岁以下）,比较组间各项指标的差异。结果哮喘组血清25-(OH)D3含量(28.14±9.34) nmol/L明显低于对照组(74.41±10.75)nmol/mL,且TIgE含量(300.01±102.13)IU/mL明显高于正常对照组(138.12±45.12)I-U/mL,哮喘组sIgE阳性检出率明显高于正常对照组,差异均有统计学意义(P<0.01)。哮喘组血清25-(OH)D3与TIgE呈负相关(r=-0.655,P<0.01),血清25-(OH)D3水平与sIgE阳性之间存在相关性(χ2=19.432,P<0.01)。儿童组sIgE阳性中食入性过敏原占25.86％,吸入性占74.14％;婴幼儿组sIgE阳性中食入性过敏原占70.21％,吸入性占29.79％;两组sIgE阳性吸入性、食入性过敏原构成有统计学差异(P<0.05)。哮喘患者血清25-(OH)D3与FEV1%pred呈正相关（r=0.568,P<0.01）。哮喘患者血清25-(OH)D3与气道阻力呈负相关（r=-0.439,P<0.01）。结论血清25-(OH)D3缺乏及不足与支气管哮喘发病存在联系,推断血清25-(OH)D3水平增高可以抑制IgE的过度表达,纠正血清25-(OH)D3水平缺乏、不足,可能改善哮喘患儿肺功能、减少过敏发生率,故补充维生素D可能成为预防和治疗支气管哮喘的一种新的有效方法。     

Defining the timing of 25(OH)D rescue following nitrogen mustard exposure

Objective: Mass exposure to alkylating agents such as nitrogen mustard (NM), whether accidental or intentional as during warfare, are known to cause systemic toxicity and severe blistering from cutaneous exposure. Thus, establishing the timing and appropriate dose of any potential drug designed to reverse or impede these toxicities is critical for wound repair and survival. Our previous data demonstrates that a single intraperitoneal injection of low-dose 25-hydroxyvitamin D3 (25(OH)D) given as early as 1?h following NM exposure is sufficient to rescue mice from pancytopenia and death. However, the duration of time following exposure where intervention is still effective as a countermeasure is unknown. In this study, we sought to assess the maximal time permissible following NM exposure where 25(OH)D still affords protection against NM-induced cutaneous injury. Additionally, we determined if a higher dose of 25(OH)D would be more efficacious at time interval where low dose 25(OH)D is no longer effective.

Methods: Low (5?ng) and high (50?ng) doses of 25(OH)D were administered intraperitoneally to mice following exposure to topical NM to assess wound resolution and survival. Mice were imaged and weighed daily to measure wound healing and to monitor systemic toxicity.

Results: We demonstrated that 5?ng 25(OH)D administered as early as 1?h and as late as 24?h post-NM exposure is able to achieve 100% recovery in mice. In contrast, intervention at and beyond 48?h of NM exposure failed to achieve full recovery and resulted in ≥60% death between days 6 and 12, demonstrating the critical nature of timely intervention with 25(OH)D at each respective dose. In order to circumvent the observed failure at >48?h exposure, we provided two consecutive doses of 5?ng or 50?ng of 25(OH)D at 48?h and 72?h post-NM exposure. Repeat dosing with 25(OH)D at 48?h and beyond led to marked improvement of lesion size with 75% recovery from mortality.

Conclusions: The opportunity to use 25(OH)D as a medical countermeasure for NM-induced toxicity has a finite of window for intervention. However, modifications such as repeat dosing can be an effective strategy to extend the intervention potential of 25(OH)D.     

Striatal and temporal cortical D2/D3 receptor occupancy by olanzapine and sertindole in vivo: a [123I]epidepride single photon emission tomography (SPET) study

RATIONALE: Previous work suggests clozapine preferentially targets limbic cortical dopamine systems, which could help account for its lack of extrapyramidal side effects (EPS) and superior therapeutic efficacy. OBJECTIVES: To test the hypothesis that olanzapine, a novel atypical antipsychotic drug, occupies temporal cortical D2/D3 receptors to a greater extent than striatal D2/D3 receptors in vivo. METHODS: Nine schizophrenic patients taking either olanzapine [(n=5; mean (SD) age: 32.5 (6.5) years; daily dose: 18.3 (2.6) mg] or sertindole [(n=4; mean (SD) age: 30.3 (7.4) years; daily dose: 16 (5.6) mg] were studied with [123I]epidepride ((S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenz amide) and single photon emission tomography (SPET). An estimate of [123I]epidepride 'specific binding' to D2/D3 receptors was obtained in patients and age-matched healthy volunteers. A summary measure was generated representing striatal and temporal cortical relative %D2/D3 receptor occupancy by antipsychotic drugs. Occupancy data were compared with previously studied groups of patients receiving typical antipsychotic drugs (n=12) and clozapine (n=10). RESULTS: Mean striatal and temporal cortical %D2/D3 receptor occupancy in olanzapine-treated patients was 41.3% (SD 17.9) and 82.8% (SD 4.2), respectively. Unexpectedly low levels of striatal relative %D2/D3 receptor occupancy were seen in two patients with typical antipsychotic-drug-induced movement disorder prior to switching to olanzapine. In the temporal cortex, mean D2/D3 dopamine receptor occupancy levels above 80% were seen for all antipsychotic drugs studied. CONCLUSIONS: The atypical antipsychotic drugs olanzapine and sertindole, in common with clozapine, demonstrate higher occupancy of temporal cortical than striatal D2/D3 dopamine receptors in vivo at clinically useful doses. This could help mediate their atypical clinical profile of therapeutic efficacy with few extrapyramidal side effects. Limbic selective blockade of D2/D3 dopamine receptors could be a common action of atypical antipsychotic drugs.     

CTGF在单侧输尿管梗阻大鼠肾组织中的表达以及活性维生素D_3干预治疗的研究

目的：观察单侧输尿管梗阻大鼠肾脏组织中CTGF的表达以及活性维生素D3（1,25-（OH）2D3）对其表达的影响。探讨1,25-（OH）2D3对大鼠肾间质纤维化的调节作用。方法：随机采用体重180~220g的清洁级Wistar大鼠,雄性,36只,随机分为假手术组（n=12）、UUO模型组（n=12）、活性维生素D3组（n=12）。造膜后活性维生素D3组给予罗盖全（骨化三醇）灌胃（3ng/100g）,假手术组及UUO模型组给予等量生理盐水灌胃。给药后第14天,28天分批（n=6）处死,取造膜侧肾脏。用HE染色及Masson染色方法观察肾脏组织纤维化程度,用免疫组化法测定结缔组织生长因子（CTGF）在各组大鼠造膜侧肾脏的表达情况。结果：与假手术组比较,UUO模型组大鼠的肾脏组织中CTGF的表达有显著增高（P〈0.05）,而在活性维生素D3治疗组中,CTGF的表达低于UUO模型组（P〈0.05）,肾间质纤维化程度也明显小于模型组（P〈0.01）。结论：CTGF在UUO大鼠肾脏中表达明显增高,1,25（OH）2D3能有效抑制UUO大鼠肾脏组织CTGF的表达,从而减轻UUO大鼠肾脏组织纤维化程度。     

Central administration of dopamine D3 receptor antisense to rat: effects on locomotion, dopamine release and [3H]spiperone binding

A 15-mer, all-phosphorothioate-modified antisense oligodeoxynucleotide (ASO) targeted against rat dopamine D3 receptor mRNA (4 μM, 5 days) significantly reduced (28%) the amount of binding sites labelled with [³H]spiperone in monolayer cultured Chinese hamster ovary (CHO) cells transfected with the complementary desoxyribonucleic acid (cDNA) for the rat D3 receptor. In contrast, D3-ASO treatment did not reduce the amount of bound [³H]spiperone in CHO cells transfected with D2 receptor cDNA. Intracerebroventricular infusion of D3-ASO (osmotic minipump, 10 μg/μl/h, 7 days) influenced dopamine receptor density in the limbic forebrain such that the upper part of the dopamine/[³H]spiperone displacement curve – tentatively representing the D3 receptor – was altered significantly. Spontaneous locomotor activity of non-habituated rats was increased significantly in D3-ASO-treated animals; in addition, in vivo microdialysis revealed a moderate increase in dopamine release in the nucleus accumbens in these animals. In all experiments, an oligodeoxynucleotide comprising the same nucleotides as the antisense sequence, but in random order, was used as control. It is concluded that the antisense strategy is useful for investigating the functional role of dopamine D3 receptors and that the dopamine D3 receptor is involved in rat locomotor behaviour. Received: 23 June 1997 / Accepted: 19 May 1998     

1,25(OH)2D3对肺炎链球菌感染的肺泡上皮细胞功能的影响

目的 观察1,25-双羟维生素D3(1,25(OH)2D3)通过miR-124-3p调控磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/AKT)通路对肺炎链球菌(SP)感染的肺泡上皮细胞功能的影响,探讨其作用机制.方法 体外培养肺泡上皮细胞A549,肺炎链球菌刺激后,随机分为10,20,40,80,160 ng·m...     

Toxicity Studies with Fluindarol, 2-[p-(trifluoromethyl)phenyl]1, 3-indandione,an Agent with Anticoagulant Properties (BS 7616 D)

The phenylindandione derivative fluindarol was subjected to a toxicological investigation in view of its possible use as an anticoagulant in man. The acute oral LD50 in rats and rabbits was 198 and 123 mg/kg, respectively. The acute intraperitoneal LD50 in the rat was 125 mg/kg. Dogs survived single oral doses of up to 2810 mg/kg; if the drug was given for four consecutive days, an oral LD50 of 118 mg/kg was obtained. In a four-week experiment in the rat, doses ranging from 108 to 7 mg/kg caused mortality which occurred sooner and more frequently in the females than the males; the organs showed haemorrhages and at the higher dose levels the liver showed parenchymal necrosis. Similar results were obtained with chlorindione, which tended to be somewhat less toxic. It was confirmed that a distinction should be made between the “toxicity” of an anticoagulant which is due to its pharmacological properties and true organ toxicity. Fluindarol was considered too toxic for clinical use.     

Regulatory Effect of 25-hydroxyvitamin D3 on Nitric Oxide Production in Activated Microglia

Microglia are activated by inflammatory and pathophysiological stimuli in neurodegenerative diseases, and activated microglia induce neuronal damage by releasing cytotoxic factors like nitric oxide (NO). Activated microglia synthesize a significant amount of vitamin D₃ in the rat brain, and vitamin D₃ has an inhibitory effect on activated microglia. To investigate the possible role of vitamin D₃ as a negative regulator of activated microglia, we examined the effect of 25-hydroxyvitamin D₃ on NO production of lipopolysaccharide (LPS)-stimulated microglia. Treatment with LPS increased the production of NO in primary cultured and BV2 microglial cells. Treatment with 25-hydroxyvitamin D₃ inhibited the generation of NO in LPS-activated primary microglia and BV2 cells. In addition to NO production, expression of 1-α-hydroxylase and the vitamin D receptor (VDR) was also upregulated in LPS-stimulated primary and BV2 microglia. When BV2 cells were transfected with 1-α-hydroxylase siRNA or VDR siRNA, the inhibitory effect of 25-hydroxyvitamin D₃ on activated BV2 cells was suppressed. 25-Hydroxyvitamin D₃ also inhibited the increased phosphorylation of p38 seen in LPS-activated BV2 cells, and this inhibition was blocked by VDR siRNA. The present study shows that 25-hydroxyvitamin D₃ inhibits NO production in LPS-activated microglia through the mediation of LPS-induced 1-α-hydroxylase. This study also shows that the inhibitory effect of 25-hydroxyvitamin D₃ on NO production might be exerted by inhibiting LPS-induced phosphorylation of p38 through the mediation of VDR signaling. These results suggest that vitamin D₃ might have an important role in the negative regulation of microglial activation.     

Dopamine receptor 3 (D3) knockout mice show regular emotional behaviour

The "Dopamine Deficiency Hypothesis" and a considerable number of recent pharmacological studies propose to thoroughly verify and improve the standard of knowledge regarding a possible role of dopamine in the pathogenesis and treatment of depression. To elucidate the potential role of D3 receptors in the development of a depressive-like phenotype, we subjected D3 receptor knockout mice to a series of selected behavioural paradigms particularly focussing on depressive-like traits. In our experimental design we exposed animals with a deficiency of the D3 receptor (D3-/-) to a standardised behavioural test battery, in which general changes of locomotion, exploration, anxiety and depressive-like characteristics (i.e. Learned Helplessness, Sucrose Consumption and Forced Swim test) can be detected. Thorough behavioural phenotyping, however, neither revealed behavioural consequences on the basal level (locomotion, exploration) nor depressive- or anxiety-like alterations. Hence, these results do not indicate an evident involvement of the D3 receptor in the development of a depression-like syndrome in mice for now. This does, however, not exclude the D3 receptor as a potential target for pharmacotherapy.     

25-羟维生素D与儿童1型糖尿病及酮症酸中毒的相关性研究

目的 探讨血清25-羟维生素D[25-(OH)D]水平与儿童1型糖尿病及酮症酸中毒(DKA)的相关性。方法 选取2006年1 月—2009年12月在本院治疗的152例患儿,其中52例为首次发病的1型糖尿病患儿,其余100例按糖尿病标准排除糖尿病。对52例1型糖尿病患儿的临床和实验室资料进行研究,分析血清25-(OH)D水平与儿童1型糖尿病及酮症酸中毒的关系。结果 1型糖尿病儿童血清25-(OH)D 的平均水平为(63.67±28.41)nmol·L^-1,显著低于非糖尿病儿童(P<0.05),差异有统计学意义。DKA患儿的血清25-(OH)D 的平均水平为(58.63±27.79)nmol·L^-1,显著低于非DKA患儿(P<0.05),差异有统计学意义。结论 血清 25-(OH)D的水平较低可能会增加儿童1型糖尿病以及发生酮症酸中毒的风险。     

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907

The selective D₃-dopamine receptor agonist 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-[N-propyl-2,3-³H]-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol ([³H]PD 128907) was used to visualise D₃-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [³H]PD 128907 has an 18- to 40-fold selectivity for D₃- over D₂-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [³H]7-OH-DPAT. [³H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [³H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D₂/D₃-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D₃-dopamine receptors in the human brain. The binding obtained with [³H]PD 128907 was qualitatively similar to that using [³H]7-OH-DPAT in the presence of GTP. However, [³H]7-OH-DPAT labelled, in contrast to [³H]PD 128907, also D₃-dopamine receptors in neocortex. The new compound [³H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D₃-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype. Received: 20 November 1995/Final version: 2 May 1996     

Synthesis of 3-(3-pyridyl)- and 3-(3-benzo[b]thienyl)-D-alanine

The DL-arylamino acid ethyl ester derivatives of β-(3-pyridyl)-DL-alanine, and β-(3-benzo[b]thienyl)-DL-alanine were synthesized by diethyl acetamidomalonate condensation with the respective arylmethyl halides followed by partial hydrolysis to the monoethyl ester and decarboxylation. Each derivative was enzymatically resolved to a separable mixture of the corresponding N-acetyl-L-amino acid and the unchanged D amino acid derivative. Acidic hydrolysis of the latter gave the corresponding D-amino acid, the optical purity of which was established by HPLC analysis of the 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) derivative. The free D amino acids were converted to D-BOC derivatives by reaction with di-tert-butyldicarbonate in tert-butyl alcohol, water and sodium hydroxide.