Samuel Natelson

Improvements in the method of separation of guanidino organic acids by column chromatography: Isolation and identification of guanidinosuccinic acid from human urine

Abstract A procedure is described for the separation and isolation of guanidino organic acid derivatives when present in low concentrations. This comprises fractionation on Dowex 50 (strongly acidic) with a series of buffers ranging upward of pH 2.2, followed by refractionation of the desired peak on Dowex 1 (strongly basic) with a sodium acetate buffer, pH 4.1. Most of the sodium ion can be removed on I.R.C. 50 (methacrylate resin carboxylic acid). The solution is then lyophilized and the guanidino organic acid derivative can be recrystallized from small volumes of water. By using this technique, guanidinosuccinic acid was isolated from the urine of a uremic patient. This is the first time that this substance has been shown to exist in biological material. The guanidinosuccinic acid was identified as the monohydrate by elemental analysis, melting point, comparison with synthetic guanidinosuccinic acid, electrophoretic mobility, paper chromatography, and by comparison of its infrared spectrum with a synthetic preparation of guanidinosuccinic acid monohydrate. The urine contained approximately 27mg per liter of guanidinosuccinic acid. This partially identifies an unidentified peak [Microchem. J. 7, 63 (1963)], which is now shown to comprise at least two guanidino derivatives; the other major component (approximately 10 mg per liter) still are not identified.

X-ray fluorescence (spectroscopy) as a tool for the analysis of submicrogram quantities of the elements in biological systems

Abstract A general discussion of the principles and applications of x-ray fluorescence analysis, also called x-ray emission spectroscopy, is described, especially as it relates to the estimation of certain elements in organic material. A typical assay procedure for hormone iodine from blood serum is described as an example of the application of this technique. The practical value of applying this technique to microgram quantities in a complex mixture for K, Ca, Cl, P, and S, as exemplified by blood serum, is discussed.

Assay for the elements chromium, manganese, iron, cobalt, copper and zinc simultaneously in human serum and sea water by X-ray spectrometry*†

Summary X-ray spectrometry can be applied for analysis of the elements important in biological systems with atomic numbers from 24–30. Human serum is ashed in platinum and the ash dissolved in dilute HCl. Sea water is evaporated to dryness and the residue is dissolved in a small quantity of dilute HCl. A mixture of acetic anhydride and glacial acetic acid is added. The amount of acetic anhydride used is calculated to react with all the water present. The sodium chloride precipitates, the chlorides of the elements assayed remaining in solution. An aliquot is evaporated to dryness in a test tube and the residue dissolved in acidified methanol. The methanol extract is evaporated on paper in a spot using a ring oven to confine the spot. The spot is then exposed to the x-ray field and scanned from 39–72 degrees 2θ. A titanium filter is used on the x-ray tube to remove chromium radiation from the tube. A flow counter is recommended to further cut down background from the white light hump. Background for each element is of the order of one cps, permitting assay of microgram quantities. In a typical experiment a pooled serum yielded values of Cr, not detectable; Mn, not detectable; Fe, 72 μg./100 ml.; Co, not detectable: Ni, 5.5 μ./100 ml.; Cu, 148 μ./100 ml.; Zn, 126 μ./100 ml. For water from Pelham Bay off Long Island Sound near shore we obtained Cr, not detectable; Mn, not detectable; Fe, 40 μ./100 ml.; Co, not detectable; Ni, 3.2 μ./100 ml.; Cu, 122 μ./100 ml.; Zn, 110 μ./100 ml. Using the ash from 5 ml. of serum or 5 ml. of sea water the limit of detectability with the present set up is 2 μ./100 ml. of the element. The Ti filter used was 0.005 in. in thickness. A thinner Ti filter (0.0013 in.) increased sensitivity by a factor of 3.4 for the elements Cr, Mn, Fe, Co and Ni. With the thinner filter the tungsten Lα lines came through and interfered with Zn and Cu analysis. An improved ring oven is described for transferring solutions to a spot on paper.

Instrumentation for the concentration of trace components of a mixture for gas chromatography: Application to the determination of acetone, ethanol, methanol and 2-propanol in blood

Summary An instrument is designed for removal of the major component of a mixture so that the minor components can be identified on the gas Chromatograph. The instrument comprises a removable small glass container seated in a constant-temperature heating block. This in turn is mounted on a vibrator or Vortex mixer for agitation. An inlet tube and an outlet tube and suitable valving permit the purging of the container with the carrying gas. For estimation of volatiles in blood, the container contains 250 mg of anhydrous copper sulfate and S g of small perforated beads. After purging the container, 100 μl of blood is injected through a rubber dam on top while the valve is turned to bypass. The container is agitated. The water is extracted by reacting with the dehydrating agent. Turning the valve to sample position permits the volatiles, other than water, to be swept into the gas Chromatograph for assay. The reaction temperature in the bottle is 50°C and in the gas Chromatograph 56°C for this procedure. With this procedure, volatiles in sewage water and other aqueous solutions can be assayed. By changing the solid reactant in the bottle other mixtures can be assayed. For example, silica gel will retain ethanol permitting trace impurities in the ethanol to be assayed.

Identification of renal calculi on micro samples by infrared analysis

Abstract A procedure is described for the microassay of renal calculi, using the infrared spectrometer, suitable for the routine clinical laboraotry. Typical spectra on both pure and mixed stones are shown. Reference spectra from 2.5–40 μ are also given for identification of the stone crystal by comparison. The spectra for the uncommon ammonium urate and calcuim acid phosphate stones from humans are also shown, and compared with authentic specimens of these crystals. The composition of the shell and core of renal calculi are analyzed and compared. The significance of this type of analysis is also discussed. The procedure is recommended as a routine procedure for the clinical laboratory because of its specificity, ease of performance, and the small sample size, ranging from 0.3 to 1 mg.

Continuous antidromic electrophoresis

Abstract An instrument is described for the continuous separation of substances with similar but not identical electrophoretic mobilities. The supporting medium (paper or plastic film supported gel) is moved continuously, mechanically, in a direction opposite to the migration of the ions. In this manner, the electrophoresis may be continued for long periods of time keeping the ions in the electrical field. Contact is made by rollers dipped into a suitable buffer. The application of the instrument to amino acids is described. Cooling is effected by passing the strip through an inert liquid (e.g., carbon tetrachloride) which is cooled by a coil through which a cooling fluid moves. The eighteen most common amino acids were readily separated. For example, leucine, isoleucine, and valine are widely separated.

Application of X-ray emission spectrometry to the estimation of the heavy elements (at. no. 79–83): Practical procedure for lead and bismuth in whole blood

Abstract A study with the X-ray spectrometer of the conditions required for the estimation of microgram quantities of the elements gold, mercury, thallium, lead, and bismuth is described. Scanning of the range from 26 ° to 38 °C (2θ) permits the resolution of the L α and L β lines of these elements. The xenon proportional counter is recommended because it has adequate sensitivity and produces a uniformly low background in this region. The molybenum target X-ray tube is used because of the absence of interfering lines. It is recommended that the sample be placed on copper or nickel supports because they exhibit low background in this range. Paper, aluminum, and silver produce a higher background. Iron also produces relatively low background. Conditions and apparatus are described for plating the element sought in a confined spot on a metal plate. Recovery of the elements in the microgram range is satisfactory for gold and mercury. Mercury, however, tends to evaporate in the X-ray field. Results with thallium, lead, and bismuth are variable but are useful for semiquantitative purposes. When dry-ashing whole blood, thallium and mercury are lost. For lead and bismuth, recovery of the elements was satisfactory, recovery ranging from 95 to 106% with a precision of ±7%. In pooled specimens, mean values of 2.3 μg/100 ml were found for bismuth and 8.8 μg/100 ml for lead in the whole blood of individuals not exposed to abnormal amounts of these elements.

Microestimation of sodium, magnesium, and barium with the X-ray spectrometer

Abstract A 0.1 mil polypropylene window on the P-10 flow counter detector permits the detection of sodium when 80 μg is placed in the X-ray field, with the chromium target tube, by X-ray spectrometry. For magnesium the limit of detection is 20 μg; for barium, 1.0 μg can be readily assayed with the tungsten tube and 0.25 μg when the chromium target tube is used. The sodium in human serum can be assayed in 50-μl samples with a reproducibility of ±7.5% (2 σ) with a 5 minute counting time. This is inadequate for routine clinical analysis. Longer counting times are impracticable for this purpose. Magnesium cannot be determined in human serum, because of its low concentration, when 50-μl is dried and placed in the X-ray field. Methods for increasing sensitivity for both these elements are proposed.

Automatic analysis of microsamples contained in capillaries with a microsample dispenser

Abstract A microsample dispenser is described for automatic microanalysis using capillaries. The accurately calibrated capillaries are placed on a turntable. The capillaries are picked up by an arm which rotates 180 ° and then lifts and tilts, to dispose the capillary in a vertical position over a receiving area. The capillary is emptied with a blast of air or by washing through with diluent. In one application the sample is applied to a top tape. This meets an intermediate porous tape (such as cellophane or micropore cellulose acetate) and a lower reagent containing tape. The three tapes go through a pressing zone. The component to be assayed, diffuses through the porous tape and reacts with a reagent to produce a stain. The stain on the lower tape is read on a densitometer. This instrument can perform at a rate of 360 determinations/hour. Application of the capillary dispenser to feed an Autoanalyzer and X-ray spectrometer are also described. The capillary dispenser can be used as an automatic autodilutor for general use or to automatically serve as a sample source for the flame photometer and atomic absorption instruments. Samples from a single capillary can be diluted and then split to serve a multiple system of analysis.

Separation of guanidino compounds by combination of electrophoresis and gel filtration. Application to human urine

Abstract An apparatus is described for preparative continuous electrophoresis. The supporting medium is a gel 24 inches by 20 inches by 1 2 inch, cooled on both sides by a flow of liquid coolant such as tap water. The buffer flows by gravity, or may be pumped downward. Electrophoresis proceeds laterally and the eluates are collected from ports at the base. A serrated block at the bottom assures even distribution of the flow through the exit ports. The electrode chambers are also cooled. Buffer in the electrode chambers, which are separated from the gel by cellophane, is constantly changed to remove salts and electrolysis products at the electrodes. Application to the separation of guanidino compounds is described with Sephadex 200 and Bio gel (acrylamide) P-10. With the instrument, it was demonstrated that a single peak obtained from uremic urine in a previous study was composed of guanidino succinic acid and guanidino acetic acids. The instrument is also generally applicable to separation of polar compounds such as polypeptides.