Clinicn Chimica Acta, 202 (1991) 191-198 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
A new immunochemical Le Phuc Thuy, L. Sweetman Department
assay for biotin * and W.L. Nyhan
of Pediatrics and The Institute of Molecular Genetics, La Jolla, CA (USA)
(Received 11 January 1991; revision received 1 July 1991; accepted 2 July 1991) Key words: Biotin; Immunochemical
Summary A double antibody technique has been developed to separate free biotin from bound biotin after competitive binding of [3H]biotin and unlabelled biotin to avidin. Antiavidin goat antibody was added followed by the addition of antigoat IgG antibody linked to agarose. Centrifugation separated the free biotin from the biotin bound to the avidin complex. The method was suitable for the detection of the amounts of biotin contained in 100-200 ~1 of plasma or 5-10 ~1 of urine. Normal values for the concentration of biotin in plasma and urine determined by this assay were 1.27 + 0.67 nmol/l and 49.1 + 35.7 pmol/mol creatinine, respectively.
Introduction In studies of inherited and acquired disorders involving biotin and its metabolites [1,2], it became of interest to determine the content of biotin in physiological fluids. It was particularly important to monitor the biotin treatment of patients with multiple carboxylase deficiency and to study the clearance of biotin in studies of the in vivo oxidation of [r3C]propionate . A new immunochemical approach was designed for this purpose. Currently most reports of the determination of biotin have been based on bioassays in which the test organism is Ochromonas danica  or Lactobacillus arabinosus 151. Several competitive protein-binding assays have been described [6,71, but their sensitivity was not good enough to permit the detection of picogram
* Present address: L. Sweetman, Childrens Hospital of Los Angeles, 4650 Sunset Boulevxd, Los Angeles, California 90027, USA. Correspondence to: L.P. Thuy, Department of Pediatrics and The Institute of Molecular Genetics, 9500 Gilman Drive, La Jolla, CA 92093, USA.
quantities of biotin in plasma. A method with suitable sensitivity required the synthesis of an iodinated biotin derivative [S-lo], which is inconvenient and time-consuming. The method described is a simple, highly sensitive assay for biotin that is suitable for use with plasma and urine. Materials
All reagents were analytical grade. [d[8,9(n)-3H]biotin, 30-60 Ci/mmol, was obtained from Amersham, Arlington Heights, Illinois. Antiavidin D goat antibody (catalogue number SP-2000, lot number 21208) was purchased from Vector Laboratories, Burlingame, California. Avidin, d-biotin, biocytin, anti-goat IgG-Agarose (catalogue number A6903, lot number 38F89451, mannitol and other chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri. Titration of avidin L3HlBiotin (43,000 DPM, 0.44 pmol, 44.3 Ci/mmol) in 20 ~1 of 0.15 mol/l NaCl was incubated with 0.16-0.80 pmol (milliunit) of avidin in 5% mannitol in phosphate buffered saline (M-PBS) in a total volume of 240 ~1. The titration was carried out as described below for the biotin assay and the percentage of [3Hjbiotin bound to avidin determined. Avidin concentrations giving 70-90% bound L3H]biotin were chosen for the biotin assay. Usually, 0.27 pmol of avidin bound approximately 70% of the t3Hjbiotin in the assay mixture. The titration of avidin was performed whenever new avidin solution was prepared or when the control showed a weak maximal binding capacity. The avidin solution containing 400 nmol/l was stable for several days at + 4 ’ C. However, the working solution of avidin (approximately 13 nmol/l) was only stable for one day on ice. Biotin assay Plasma (200 ~11, urine (5-10 ~1) or biotin standard (25-300 pg) was mixed with i3H]biotin (43,ooO DPM, 0.44 pmol in 20 ~1 0.15 mol/l NaCl) prior to addition of avidin (usually 0.27 pmol in 20 ~1 of M-PBS) in the total volume brought to 240 ~1 with M-PBS. The mixture was shaken for 30 min. Ten ~1 of antiavidin antibody (2 mg) was added and the tubes were shaken for 30 min. Fifty ~1 of a suspension of antigoat IgG-agarose (25 ~1 packed gel) was added and the mixture was again shaken for 60 min followed by centrifugation at 10,000 X g for 10 minutes in a Beckman Microfuge, Model 12 (Beckman Instruments, Inc., Palo AIto, California). The whole assay was carried out at room temperature. The activity of the isotope of free [3H]biotin in 150 ~1 of supernatant fluid was determined in a Beckman Liquid Scintillation Counter, Model LS 3801 (Beckman). Siliconized microfuge tubes were used, and the assay was carried out at room temperature. A blank ttibe containing no avidin was used to monitor the overall recovery of i3Hlbiotin. A recovery > 80% was acceptable. Another tube containing no unlabelled biotin was
used to check the maximal binding capacity of avidin. The percentage of bound [3H]biotin was calculated and plotted against the concentration of biotin in standard solutions on semi-log paper. Biotin contents of unknown samples were assessed from the standard curve. Results
The standard curve obtained with 25-300 pg of biotin (0.10-1.25 pmol) per assay is shown in Fig. 1. The standard curve was linear from 0 to 200 pg of biotin. The standard curve as drawn in Fig. 1 was useful for the estimation of up to 300 pg biotin/assay. Biotin content was determined in 10 identical samples of diluted normal urine. The mean value and standard deviation were 9.96 + 0.39 pmol/mol creatinine and the coefficient of variation was 3.91. These results indicated that the assay was highly precise. A quadruplicate of the same sample was assayed for biotin on two other occasions using two different standard curves. The mean values and standard deviations were 10.83 + 1.72 and 10.55 IL 0.69 ~mol/mol creatinine indicating that the method was reliable and the variation between runs small. The average content of biotin in 200 ~1 plasma or 5 /..~ul urine were 62 pg and 190 pg, respectively, values which were in the sensitive range of the standard curve. For the detection of biotin in plasma and/or urine of patients receiving pharmacologic doses of biotin, a standard curve of 250-3000 pg per assay was established. This
( pg 1
Fig. 1. Standard curve for the assay for biotin. Unlabelled biotin was incubated with [3H]biotin and avidin. The separation of bound and unbound [3H]biotin was achieved by the addition of goat antiavidin antibody and anti-goat IgG antibody as described in ‘Materials and Methods’ section.
194 TABLE I Biotin recovery from plasma Total biotin recovered (Pg) 40
Biotin added (PFS) None
50 50 50
50 100 200
91 140 235
114 100 97.5
required scaling the reagent concentrations up 10 times, but the total volume remained the same at 240 ~1. A linear relationship was observed between plasma and the picograms of biotin detected. Thirty, 70, 115, and 150 pg of biotin were detected in 50, 1.00, 150 and 200 ~1 of plasma, respectively. A similar linear relationship was shown when volumes of urine were increased. Sixty, 112, 160 and 214 picograms of biotin was detected in 1, 2, 3, and 4 ~1 of urine, respectively. The effects of biotin added to 50 ~1 of plasma are shown in Table I. When 50-200 pg of biotin was added to plasma prior to the assay, the recovery of added biotin was 97.5 to 114%. The recovery of biotin added to 1.25 ~1 of urine was 85-105% (Table II). Assay with this method of plasma obtained from 14 healthy adults revealed a mean concentration of biotin of 1.27 + 0.67 nmol/l; the range was 0.30-2.77 nmol/l. Normal values for urine were a mean concentration of 0.156 _t 0.070 pmol/l (n = 16) and a range of 0.078-0.332 pmol/l. Expressed in relation to creatinine content the mean value was 49.1 + 35.7 pmol/mol creatinine (range 9.7-119.0). There was little influence of anticoagulants on the assay. In a paired experiment, a sample of plasma treated with heparin and with EDTA gave values of 1.07 nmol/l and 1.23 nmol/l, respectively. It has been thought that biotin might bind to plasma proteins. However, we found that all of the biotin or biotin derivatives which were able to bind to avidin could be found in a protein-free filtrate of plasma. Plasma was filtered through the Centrifree micropartition system (Amicon Corporation) by centrifugation at 1,000 x g for 30 min in a fixed-angle centrifuge. This membrane has a 30,000 Da cutoff. The biotin contents of whole plasma and filtrate were determined to be 2.01
TABLE II Biotin recovery from urine Urine (CL0 1.25 1.25 1.25 1.25
Biotin added (PP) None 50 100 200
Total biotin recovered (Pg) 45 90 130 255
% Recovery _ 90 85 105
nmol/l and 2.09 nmol/l, respectively. Furthermore, when 0.7 pmol of i3H]biotin (47,000 DPM) was incubated with 500 ~1 plasma for 3 h at room temperature followed by filtration through the Centrifree system, 92% of [3H]biotin was recovered in the filtrate. The filter has been described to retain > 99.9% serum protein and < 5% thyroxine. An 8% loss of [3H]biotin through the entire experiment might represent nonspecific absorption to the membrane and/or experimental error, rather than binding of radioactive biotin to plasma proteins, in view of the results obtained from the whole and filtered plasma. The high renal elimination rate of biotin would be consistent with the fact that biotin is free in plasma. Similar experiments showed that urine contains free biotin, not protein bound biotin. Discussion Results for the biotin content of plasma agreed well with the value of 1.26 rf: 0.50 nmol/l published by Horsburgh and Gompertz  who used an iodinated biotin conjugate in a competitive protein binding assay. The data from bioassays have ranged from 0.4 to 2.0 nmol/l for the biotin concentration of plasma [4,5]. Compounds such as aspartic and oleic acid have been observed to act like biotin in bioassays . Sanghvi et al.  recently obtained a value of 2.13 f 0.90 nmol/l biotin in plasma an assay in which nitrocellulose membrane was employed to separate avidin from biotin. Livaniott et al. reported that the mean biotin content in plasma was 1.39 nmol/l (range 0.4-3.44 nmol/l) [lo]. Their results for the biotin content of urine (118 f 66 ng/mg creatinine) were comparable to those observed in this study. Biotinidase is highly active in plasma. Thus normal plasma would be expected to contain no biocytin. However, it was postulated that plasma and urine of patients with biotinidase deficiency might contain biocytin. This has been reported by Bonjour et al. [ll] who postulated that this compound may account for some of the late toxicity of biotinidase deficiency. It has also been postulated to compete with biotin for renal tubular reabsorption of biotin . Biocytin competes more weakly with [3H]biotin for binding to avidin than does unlabelled biotin and [3H]biotin. Figure 2 indicates that a given amount of biocytin gives only 50% of the binding efficiency as does the same amount of biotin. Therefore, in specimens obtained from patients with late-onset multiple carboxylase deficiency it is important to separate biotin from biocytin prior to the assay of biotin using [3H]biotin or biocytin using [ 3H]biocytin. Biotin assays based on the competitive binding of [3H]biotin and unlabelled biotin to avidin have been described [6,7]. However, there were two major problems. The most important was that these methods were not sensitive enough to detect biotin at the concentrations that it is found in plasma in which it is in the pmol/ml range. This is an especially important consideration for studies in pediatric patients in whom amounts of plasma available are limited. In the second place, the separation of free biotin from the biotin-avidin complex by filtration through nitrocellulose membrane , precipitation by ZnSO,  or absorption on
0.6 Bioiin of Biocylin
( picomole 1
Fig. 2. Standard curves for the assay for biotin (01 and for biocytin (0) using [3H&iotin as radioligand. Biotin (0.10-1.23 pmol) or biocytin (0.13-0.81 pmol) was incubated with 1~H~iotin and the assays were carried out as described in ‘Materiais and Methods’ section.
bentonite  was unsatisfactory when femtomoles of avidin were used in order to improve the sensitivity of the assay. In our method these problems were overcome by the use of f3Hlbiotin of very high specific activity (30-50 Ci/mmol) and a double antibody technique to separate the biotin-avidin complex from free biotin. Antiavidin antibody raised in goat bound to biotin-avidin forming a tertiary complex (biotin-avidin-antiavidin antibody). A second antibody, antigoat IgG antibody raised in rabbit and linked to agarose, provided a solid phase to pull out the tertiary complex from the liquid phase. Then a simple centrifugation was sufficient to separate the bound and free biotin mole&es. To prevent the loss of avidin by nonspecific absorption, silicunized tubes and mannitol have been used. Since the binding of biotin to avidin is practically irreversible, it was crucial to mix well [3H]biotin and unlabelled biotin before the addition of avidin, thus assuring a fair competition between these biotin molecules. Antibiotin antibody was commercially available. However the titer of this antibody was not high enough to be used in a direct radioimmunoassay for biotin (unpubl. data). It is concluded that the biotin assay developed is simple and sensitive. The range of sensitivity from 25 to 300 pg/assay has permitted the quantification of biotin of small samples of plasma, as well as urine. Therefore, it is suitable for studies in pediatric patients. The method has been used in our iaboratory to monitor the course of treatment with biotin in patients with multiple carboxylase
deficiency and to assess for nutritional deficiency of biotin. The method is also suitable for the study of the clearance and absorption of biotin. Furthermore, the method has been useful in the location of fractions containing biotinylated proteins during purification of biotin-containing carboxylase enzymes. Acknowledgements This research was aided by USPHS grants from the Maternal and Child Health Bureau, No. MCJ 004007 and the National Institute of Child Health and Human Development, No. HD 18789, and the Allen Foundation, Midland Michigan. References 1 Nyhan WL, Sakati NO. Diagnostic Recognition of Genetic Disease. Philadelphia: Lea & Febiger, 1987;50-65. 2 Sweetman L, Surh L, Baker H, Peterson RM, Nyhan WL. Clinical and metabolic abnormalities in a boy with dietary deficiency of biotin. Pediatrics 1981;68:553-558. 3 Barshop BA, Yoshida I, Ajami A, Sweetman L, Wolff JA, Sweetman FR, Prodanos C, Smith M, Nyhan WL. Metabolism of l-13C-propionate in vivo in patients with disease of propionate metabolism. Pediatr Res 1991;30:15-22. 4 Baker H, Frank 0, Matovich VB, et al. A new assay method for biotin in blood, serum, urine and tissue. Anal Biochem 1962;3:31-39. 5 Wright LD, Skeggs HR. Determination of biotin with Lactobacih arabinosus. Proc Sot Exp Biol Med 1944;56:95-98. 6 Sanghvi RS, Lemons RM, Baker H, Thoene JG. A simple method for determination of plasma and urinary biotin. Clin Chim Acta 1982;124:85-90. 7 Bhullar RP, Lie SH, Dakshinamurti K. Isotope dilution assay for biotin. Ann NY Acad Sci 1985;447:122-128. 8 Horsburgh T, Gompertz D. A protein-binding assay for measurement of biotin in physiological fluids. Clin Chim Acta 1978;82:215-223. 9 Wai Chan P, Bartlett K. A new solid-phase assay for biotin and biocytin and its application to the study of patients with biotinidase deficiency. Clin Chim Acta 1986;159:185-196. 10 Livaniou E, Evangelatos GP, Ithakissios DS. Biotin radioligand assay with an iz51-labeled biotin derivative, avidin, and avidin double-antibody reagents. Clin Chem 1987;33/11:1983-1988. 11 Bonjour JP, Bausch J, Sourmala T, Baumgartner ER. Detection of biocytin in urine of children with congenital biotinidase deficiency. Int J Vitamin Nutr Res 1984;54:223-231. 12 Baumgartner R, Sourmala T, Wick H, Bausch J, Bonjour JP. Biotinidase deficiency: factors responsible for increased biotin requirement. J Inherit Metab Dis 1985;8(Suppl 1):59-64. 13 Hood RS. Isotopic dilution assay for biotin: use of [i4C]biotin. Methods Enzymol 1979;62:279-283.