THE MAN AND THE METHOD



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 Medical 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 biological 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 pancreatic 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.

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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 these 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 hospitals 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, thermal burns, extended use of phosphate-binding antacids and respiratory alkalosis.

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 increase 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.

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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, other 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 available 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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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

MgHPO4 0.1 0.03 2

Free 3.1 1 85

H2PO4 0.6 0.2 17

HPO4 2.5 9.8 68

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

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 phosphate 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)2D3

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

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