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THE MAN AND THE METHOD

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					                                 THE MAN AND THE METHOD

                                      Suman Kapur and S P K Gupta


         American author Doron Antrim has said, "You've probably never heard of Dr. Yellapragada
SubbaRow. Yet because he lived you may be alive and are well today. Because he lived you may live
longer." This is certainly not an overstatement. His basic contributions towards isolation,
characterization and delineation of the biological effects of several life -saving and life - prolonging
vitamins, antibiotics, anti-cancer drugs have stood the test of medical practice for more than half a
century. Just this year, on 26th January, 1998, World Health Organization declared that a single dose of
Dr. SubbaRow's diethlcarbamazine (DEC), administered concurrently with ivermectin, will keep blood
free of filarial worms for a year.

         Dr. SubbaRow of Bhimavaram, Andhra Pradesh, India wanted as a young man to seek
knowledge about the relation of man to God. As per his personal logs, the Ramakrishna Mission
however motivated him to study medicine. In his final year at Madras Medica l College, he met a young
American doctor who guided him to Harvard's School of Tropical Medicine (HSTM). He was indeed
accepted by HSTM in 1921 but the death of his elder brother, who was to secure him a scholarship for
studies in USA, frustrated Dr. SubbaRow's plans that year. He could leave for Harvard two years later
when the Malladi Satyalinga Naicker Charities promised the scholarship. He was then 28 years of age.

         During the next 25 years, Dr. SubbaRow devoted himself in USA to relentless toils in bi ological
and medical research. Moving from HSTM to Harvard Medical School (HMS), he devised a test for the
estimation of phosphorus in body fluids, co-discovered phosphocreatine and ATP and worked extensively
on understanding the biology of vitamins. After moving in 1940 to Lederle Laboratories, he directed
research that yielded folic acid, tetracycline, methotrexate and DEC.


        Herein we review Dr. SubbaRow's first scientific paper, "The colorimetric determination of
phosphorus", and its implication and application in present-day medicine, 73 years after its publication in
the Journal of Biological Chemistry', in 1925.


PHOSPHORUS IN HEALTH AND DISEASE

         Phosphorus is an essential element of living matter. It is found in every cell of the body and is a
major constituent of the skeletal system, a central part of energy transfer in cellular biochemical reaction
and an important component of membranes and other cell structures. Thus, the maintenance of phosphorus
balance with serum inorganic phosphorus concentration within normal range is critical -for the normal
function of the organism. In general the intestine is the only organ through which exogenous phosphate is
added to the body and the kidney is the only organ through which excess phosphate is excreted. Thus ,
regulation of phosphate fluxes in these two organs is most important in the regulation of phosphorus
balance. Processes that disturb the regulatory system responsible for the maintenance of phosphorus
balance cause significant clinical problems.

DISTRIBUTION AND BALANCE
         The human body contains approximately 600 to 700g (17 mole) of phosphorus. Some 85 to 87%
of this is combined with calcium in bones and teeth. About 10% of total phosphorus is combined with
proteins, lipids, carbohydrates and other macromolecules and resides in the soft tissues intracellularly.
Intracellular phosphorus is organic phosphate and present as an integral constituent of phospholipids,
nucleic acids and phosphoproteins which are essential for maintenance of cellular integrity and metabolic
functions. About 10% of total phosphorus exists in the inorganic state and is widely distributed in various
chemical compounds. The amount of inorganic phosphate in the cell is small but it is very critical for cell
function as this is the only form utilised for the synthesis of adenosine triphosphate (ATP). Animal studies
have shown that approximately 3 gm of the stored bone phosphorus is exchangeable with the extracellular
fluids. In pathological conditions, a substantial amount of phosphorus may leave the skeleton, leading to
demineralisation or loss of whole bone mineral. Severe phosphorus depletion results in net release of
phosphorus as well as calcium for bone regardless of the need for calcium in the extracelluar fluids.
         The External balance of phosphorus must be zero in healthy adults when it is averaged over an
appropriate period of time. Over the first 20 years of life, the phosphorus balance is continuously positive,
with an average of 2-3 mmol per day. From the fourth to fifth decade of life, the external balance in normal
healthy subjects becomes negative, with an average loss of approximately 1 mmol phosphorus \day due to
involutional bone loss.

DIETARY REQUIREMENTS AND SOURCES

         Phosphorus is present in nearly all foods. Consequently a dietary deficiency is not known to
occur in man. Dietary phosphate, ranging from 32-64 mmol/day (nearly 1.5 g), is in both inorganic and
organic forms. Major portions of the organic form are also hydrolyzed to inorganic phosphate and
primarily absorbed in the duodenum and jejunum. Ileum and colon can also absorb smaller amounts of
phosphate. When dietary phosphate supply is low, faecal phosphorus also decreases proportionately.
Endogenous sources of phosphorus are the saliva, the gastric, intestinal and p ancreatic secretions and the
debris of enterocytes. A portion of endogenous phosphate is absorbed and nearly 100 -300 mg is excreted
daily in the faeces. Faecal phosphorus is present in both inorganic and organic forms. Urinary phosphate
excretion also decreases in response to dietary restriction of phosphorus.

         The mechanism by which phosphate enters or exits cells has only recently been elucidated.
Na+-Pi co-transporter has been cloned and characterized. Once inside the cytosol, the phosphate anion
participates in various phosphorylation reactions in the cytosol, is transported in to the mitochondria or
exits the cell across the basolateral membrane. Intracellular increase in concentration of cyclic AMP
(cAMP) lead to an altered rate of the Na+-P' co-transporter. Several physiological mechanisms regulate
transport of phosphate by the cell members. These cause gain or losses of phosphate by the cells with
reciprocal changes in the plasma phosphate concentration. Since inorganic phosphate ion concentrations
in the intracellular and extracellular compartments are in equilibrium, the shifts of inorganic phosphate
across cell membranes involve a transient change in intracellular organic phosphate compounds, such as
glucose-6-phosphate, ATP and phosphocreatine. Agents like insulin, glucose and fructose and changes
in blood pH, cause a transfer of phosphate from the plasma to the cells and lead to a transient fall in the
plasma phosphate concentration.

                                   Table 1-Composition of plasma phosphorus
                    Composition                       mg/dL                  mmo1/L            %
           Total Phosphorus                              12.1                   3.9             -
           Acid-insoluble phospholipids                    8.1                  2.6             -
           Acid-soluble                                    0.3                   0.1            -
           Organic ester                                    3.7                  1.2             -

           Inorganic                                        0.01                 0.003              -

           Pyrophosphate orthophosphate                    3.7                    1.2               100

           Protein bound                                    0.4                   0.1                10

           Complexed                                        0.2                   0.07                  5

           CaHPO4                                          0.1                   0.04                   3

           MgHPO 4                                         0.1                   0.03                   2

           Free                                            3.1                   1                  85

           H 2PO4                                          0.6                   0.2                17

           HPO 4                                          2.5                    9.8            68
The normal levels of phosphorus in plasma range between 2.5 and 4.8 mg/dl. The levels are 25 -50%
higher in growing children. Composition of plasma phosphorus can be seen in Table 1.

REGULATION OF PLASMA PHOSPHORUS CONCENTRATION
         Close endocrine regulation of absorption and excretion of phosphorus maintains steady state
plasma phosphorus levels. Hence, intestine and kidney are the two major organs that determine the
external balance and plasma concentration of phosphorus. Intestinal absorption of phosphorus
occasionally floods the extracellular fluids, the kidneys maintain homeostasis by excreting the precise
amount absorbed in the excess of the body's need. The reabsorbtion of phosphorus in the renal tubules is
controlled by a multitude of hormonal, metabolic and dietary factors. Of thes e parathyroid hormone
(PTH) and dietary intake of phosphate are the major determinants of renal tubular phosphates is
reabsorbtion. Normally, 85-90% of filtered phosphate is reabsorbed. Table 2 lists the factors that increase
or decrease the tubular phosphate transport.

CLINICAL DISORDERS ASSOCIATED WITH PHOSPHORUS
Hypophosphatemia

Transient hypophosphatemia induced by any agent is harmless. Lowered plasma phosphorus is seen in
several clinical conditions (Table 3). Upto 3.1 % patients admitted to hospita ls show lower plasma
phosphorus. However, under certain circumstances, it results in significant clinical consequences and can
lead to morbidity and mortality. Some patients may even suffer from stupor, coma and grand mal
seizures. Hypophosphatemia is not always associated with phosphate depletion, and is mostly transient.
Phosphate depletion, on the other hand, causes severe hypophosphatemia with plasma levels below 1.0
mg/dL. Severe forms are seen in diabetic ketosis, hyperalimentation, alcoholism, therma l burns, extended
use of phosphate-binding antacids and respiratory alkalosis.

               Table 2 - Factors affecting Tubular Phosphate Transport

                             A. Factors that decrease Phosphate Reabsorption
       Parathyroid hormone
       High dietary phosphate intake
       Extra-cellular fluid volume expansion
       Diuretics
       Calcitonin
       Glucocorticoids
       Thyroid hormone
       Alcoholism
       Urinary alkalization
                                B. Factors that increase phosphate reabsorbtion
       Dietary restriction of phosphate
       Insulin
       Hyperealcemia
       Growth hormone
       Vitamin D metabolites

         Considering the importance of phosphate as a source of energy, the fact that virtually all organ
systems are affected in severe hypophosphatemia and phosphate depletion is not surprising. Table 4 lists
the manifestations of phosphate depletion on various organ systems. Several commercial preparations are
available today which can be used as replacement therapy for hypophosphatemia. Table 5 lists some of
these preparations. Care needs to be practiced in administering phosphates as it may lead to diarrhea,
hypocalcaemia, metabolic acidosis and hyperphosphatemia.

HYPERPHOSPHATEMIA
A change in the renal function and threshold for phosphates can lead to an increa se in
hyperphosphatemia due to a decrease in its urinary excretion. Some of the factors causing
hyperphosphatemia and the associated clinical symptoms are summarized in Table 6. Clinically
significant effects include band keratopathy, conjunctival hyperaemia, hypoxia, renal failure and
nephrocalcinosis. Treatment modalities for hyperphosphatemia include dialysis, dietary restrictions and use
of intestinal phosphate binders.




                     Table 3-Clinical disorders associated with Hypophosphatemia
     Increased shift of phosphate into cells and bone
             Carbohydrate load
             Hyperalimentation and nutrition recovery syndrome*
             Respiratory alkalosis*
             Androgen therapy
             Rapid cell growth*
             Gram negative sepsis
             Renal phosphate loss
             Hyperparathyroidism
             Renal tubular disorders, primary and secondary
             Volume expansion
             Diuretic therapy
             Hypomagnesemia
             Corticosteroid administration
             Sodium bicarbonate infusion

     Decreased intestinal absorption of phosphate and/or increased intestinal loss of phosphate
             Selective dietary phosphate deficiency*
             Administration of phosphate-binding antacids*
             Vomiting
             Various malabsorbtion syndromes

     Combination of increased shift of phosphate into cells and bone with renal phosphate loss
            Metabolic acidosis
            Diabetic ketoacidosis*
            Severe third degree burns*
            Gout
            Sodium lactate administration

     Combination of renal phosphate loss and decreased intestinal absorbtion of phosphate
            Rickets/osteomalacia
            Renal tubular disorders associated with altered vitamin D metabolism
            Malabsoi'ption
            Hemodialysis*

     Combination of renal phosphate loss, decreased intestinal absorbtion of phosphate and
     increased shift of phosphate into cells and bone
             Alcoholism*
             Postrenal transplantation

     *Conditions that may result in severe hypophosphatemia and manifestations of phosphate
     depletion syndrome.
  Table 4-Clinical and biochemical manifestation of hypophosphatemia and phosph ate
                                        depletion
  Serum biochemistry
     Hypophosphatemia
     Normal or hypornagnesernia
     Normal; hepercalcemia, or hypocalec;mia
     Acid-base disturbances
  Hematalogic
       Red blood cell rigidity, hemolysis
       Reduced 2,3-diphosphoglyceride content in red blood cell (tissue hypoxia)
       Impaired phagocytosis
       Platelet dysfunction
  Gastrointestinal
       Alterations in calcium, phosphorus, and magnesium absorption
  Hepatic
       Excerbates abnormal liver function
  Pulmonary
       Hyperventilation
       Hypoventilation
  Cardiac
       Myocardial dysfunction
       Cardiomyopathy
  Muscular
       Weakness
       Myopathy
       Rhabdomyolysis
  Skeletal
       Bone pain
       Pseudofractures
       Osteomalacia/rickets
       Bone cell dysfunction
       Decreased bone calcium, phosphorus, and magnesium
   Renal
       Hypophosphaturia
       Hypercalciuria
       Hypermagnesuria
       Bicarbonaturia
       Increased synthesis of 1,25 (OH) 2 D 3
    Central nervous system
       Neuroencephalopathy

                  Table 5-Commercially available phosphate preparations
                         For 1 g        with l g        with l g
                         Phosphorus     Na              K
                                        [mEq)           (mEq)
For intravenous use
In-Phos                  40.0ml         6.5             8
Hyper-Phos-K             15.            0               0.5

For oral use
Fleet's Phospho           6.2ml               57       0
Neutra-Phos               300ml (or 4cap)    28.5      28.5
Neutra-Phos-K             300ml(or 4cap)      0        57
Phos-Tabs                 6 Tab                0       57
K-Phos M.F.               8 tab             2.9        1.14
K-Phos No.2               4 tab             5.83       2:25
K-Phos Neutral             4 tab            5.83       2.25
K-phos Original            7 tab            0          3.67
K-Phos Alkaline            4 tab            13.9       2.3

Abbreviation : tab-tablet; cap-capsule.
                                     Table 6-Evaluation of hyperphosphatemia
         Serum Phasphate>5mg/dL
         Renal failure : acute and chronic
            GFR>25-30m1/min
         Phosphate loading
            Exogenous
                  Oral
                  Enema
                  Intravenous
                  Skin
            Endogenous
                  Tissue breakdown
                  Rhabdomyolysis
                  Cytotoxic therapy for leukemias and lymphoma
                  Acidosis
          Increased TmP/GFR
                  Hypoparathyroidism
                  Pseudohypoparathyroidism
                  Hyperthyroidism
                  Tumoral calcinosis
                  Growth hormone excess

         Abbreviations : GFR, glomerular filtration rate; TmP, maximum tubular reabsorptive rate for
         phosphate



         Hence the importance of phosphorus determination in body fluids in the biochemical laboratory as a
diagnostic tool. In fact, phosphorus determination is one of the first procedures taught in biochemistry course.


DETERMINATION OF INORGANIC PHOSPHATE
            Diabetics and renal failure patients are routinely found to have imbalanced phosphate and receive
parenteral treatment at either remove excess or makeup deficient phosphate in their biological fluids. In the
clinic, one often encounters patients with metabolic acidosis. In these patients estimation of phosphorus levels,
affirming hyperphosphatemia, not only aids diagnosis, but also decides the course of treatment and clinical
management. The first accurate estimations of inorganic and organic phosphates were done by Fiske and
SubbaRow in 1925.
            SubbaRow had gone to USA in 1923 to find cures then unknown to modern medicine for major
  killer diseases. But at the end of a year at HSTM, the promised Malladi scholarship materialized. As that
  was not tenable for medical studies, he enrolled himself for a summer course in Biochemistry at the
  Harvard Medical School (HMS). "My ambition", he wrote home, "is to study biochemistry which deals
  mostly with the normal chemical processes of the body".
            Prof. Otto Folin, head of Biochemistry at HMS, assigned Dr. SubbaRow to Cyrus Hartwell Fiske
  and set him on the job of resolving problems associated with the method for estimation of inorganic
  phosphate worked out four years earlier in his department by Prof. R. D. Bell and Dr/ E. A, Doisy. The
  method involved the conversion of tissue phosphorus into phospho -molybdic acid and breaking it down
  with hydroquinone into a blue substance that lent itself to measurement by color comparison. However,
  the blue color of the end product faded too rapidly, in a rather irregular fashion, making its reliable
  measurement difficult. A.P.Briggs, an associate of Dr.Bell, made some modifications but these slowed
  the reaction significantly. And, during the extended reaction time, othe r substances interfered in the
  development of colour, resulting in an unacceptable error margin of 4 -10%.
            The task set for Dr.SubbaRow was to find a reducing agent that would in low concentrations
  completely break down phospho-molybdic acid within a short period. This reducing agent should not be
  affected by trichloroacetic acid, another component of the reaction , and should not require sulphuric acid
  to digest phosphorus.
          The first success came when SubbaRow discovered that 5-aminosaligenin as a reducing agent
gave in 30 min 20% more color than hydroquinone. But, the target was complete color development in less
than 5 min. He continued to experiment with newer reducing agents until 1,2,6 -aminonapthol sulphonic
acid, ANSA, was found to be 50 time more active than hydroquinone and the color development took less
than a minute. This reagent, however, was difficult to prepare. Luckily SubbaRow found its isomer, 1,2,4 -
ANSA, to work equally well, easy to prepare in the laboratory and, moreover, readily availabl e in the
market due to its application in dye manufacturing. 1,2,4-acid, as it was known in the trade, stood every
test. The old ammonium molybdate of Bell and Doisy served to convert phosphorus into phospho -
molybdic acid before ANSA was added to get the blue end product which absorbs light at 340 nm and is
measure by colorimetry. It gave accurate readings even in the presence of ten times more inhibiting
material than permissible with hydroquinone. After extensive standardization and analysis, SubbaRow
recorded proudly that the assay "is correct to 1/100,000th of a grain". Midway through all this, Folin ended
SubbaRow's probation and accepted him as a regular graduate student along with the job of a night
assistant in HMS Library.
         Assigned the task in the summer of 1924, as a novice in biochemistry, SubbaRow had been able-
by working in the lab from 8 in the morning till well past midnight with only brief breaks for food -to
resolve all problems by Fall so much so his hard-to-please supervisor Fiske told a colleague: "That man!
His analyses are right on the dot!"
         The word of success soon got around and the American Society of the Biological Chemists invited
Fiske and SubbaRow to demonstrate the new phosphorus method before its annual meeting on December
29, 1924. The Society approved the method and the 1925 editions of biochemistry textbooks carried
descriptions of the "Fiske-SubbaRow Method". It is courtesy in research that the name of both the worker
and his supervisor is ascribed to the work or findings. Additionally, the name of the senior associate, one
with a reputation like Dr. Fiske's would gain ready acceptance than if it only bore the name of a novice as
yet unknown to the scientific community. Dr. Fiske also wrote up the paper that was published at the end
of a year in the December 1925 issue of the Journal of Biological Chemistry.
         In 1930, Kuttner and Liechtenstein', increased the sensitivity of the method by using stannous
chloride as the reducing agent. And in 1964, Hurst improved the performance of stannous chloride by
inclusion of hydrazine, which stabilized the blue colored complex further. Current methods include ferrous
sulphate as the reducing agent, a modification introduced in 1944 by summer' to enable the reaction to be
carried out in a weakly acid solution and provide greater specificity with mixture of inorganic phosphorus
and labile esters; and the final color also develops more rapidly and remains stable for at least 2 hr.
         Second generation methods use phosphomolybdovanate or molybdic acid -malachite green but the
choice remains the reduction of phosphomolybdate finessed originally by SubbaRow under the supervision
of Fiske. No wonder succeeding generations of biochemist have known the procedure, despite modification
over the years, as the "FiskeSubbaRow Method".


CONCLUSION
         Fiske and SubbaRow used their phosphorus method to co-discover phosphocreatine and ATP and
to isolate a number of phosphorus compounds in liver, spleen, pancreas and kidneys as well as nucleotides
involved in the synthesis of RNA.
         According to Nobel Laureate George H Hitchings, who was SubbaRow's Colleague as a graduate
assistant of Fiske, some of the nucleotides isolated by SubbaRow had to be rediscovered years later by
other workers because Fiske, undergoing a change in personality, would not let the results see the light of
publication. Progress of science in the field of nucleic acids was to that extent delayed.
         This and SubbaRow's penchant for pushing to limelight the outstanding youngmen in research
teams he directed at Lederle Laboratories, rather than himself as director, are the reasons why his name is
cited in scientific literature only in association with a procedure he worked out as a novitiate in
biochemistry.
         SubbaRow thought he would honor the conditions of the Malladi Scholarship by working in a
field of no medical significance. And yet his labours, keeping body and soul together with meagre
scholarship funds, yielded a procedure that has become an important tool for diagnosis of metabolic
diseases. One can only conclude that whatever SubbaRow touched in a quarter century of dedicated
research life was destined to become significant in humankind's, continuing fight against disease.

ACKNOWLEDGEMENT
Grateful acknowledgements to Ms.Shoma Gomes for help in conducing part of the literature search and
Dr.Yamini Mailapur for clinical case studies.

REFERENCES

    1.   S P K GUPTA AND E L MILFORD: IN QUESG OF PANACEA (EVELYN PUBLIOSHERS, NEW
         DELHI, 1987)
    2.   C H FISKE AND Y SUBBAROW: J BIOL CHEMISTRY 66 (1925) 375
    3.   T KUTTNER & L LIECHTENSTEIN: J BIOL CHEM 86 (1930) 671
    4.   R D HURST: CAN J BIOCHEM 42 (1964) 287
    5.   J B SUMMER: SCIENCE 100 (1944) 413
(FROM INDIAN JOURNAL OF EXPERIMENTAL BIOLOGY 36 (1998) 1087-1092)

				
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